Synthesis and methods of use of new antimitotic agents

ABSTRACT

Oxidative cyclization of bis-naphthyl ethers allows concise total syntheses of palmarumycin CP, and deoxypreussomerin A in 8-9 steps and 15-35% overall yield from 5-hydroxy-8-methoxy-1 -tetralone. A small library of palmarumycin analogs was created. Biological evaluation of these naphthoquinone spiroketals against MCF-7 and MDA-MB-23 1 human breast cancer cells revealed several low-micromolar growth inhibitors. A number of the analogs inhibit the thioredoxin -thioredoxin reductase system.

SYNTHESES AND METHODS OF USE OF NEW ANTIMITOTIC AGENTS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/219,282 filed Jul. 19, 2000.

[0002] Statement Re2arding Federally-Sponsored Research This inventionwas supported by the United States Government under Grant No. CA-78039awarded by the National Institutes of Health. The Government may havecertain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates in general to anticancer agents,and more specifically to synthetic analogs of deoxypreussomerin,palmarumycin CPI and related naphthoquinone spiroketals, which exhibitantimitotic activity.

BACKGROUND OF THE INVENTION

[0004] The cell cycle consists of series of stages abbreviated Gi - S -G2 - M. GI stands for gap number 1; S for synthesis (DNA synthesisoccurs); G2 for gap number 2 and M for mitosis or cell division. Controlof cell division is very complex and involves regulation at a number oflevels. In cancerous cells, the normal regulatory processes are somehowdisrupted and cell growth is uncontrolled.

[0005] Tubulin is a protein that polymerizes into long chains orfilaments that form microtubules. Microtubules are hollow fibers, whichserve as kind of a skeletal system for living cells. Microtubules havethe ability to shift through various formations, thus allowing the cellto undergo mitosis. The formation-shifting of microtubules is madepossible by the flexibility of tubulin monomers, especially in thepresence of agents/drugs and proteins that bind tubulin.

[0006] Interest in tubulin has increased recently because a naturalsubstance (paclitaxel) found in the bark of the Pacific yew tree, wasshown in clinical tests to be an effective treatment for a number ofcancers including ovarian, breast, and lung. Paclitaxel prevents celldivision by promoting the assembly of and inhibiting the disassembly ofmicrotubules.

[0007] Cell cycle checkpoints are critical regulators of genomeintegrity and faithful cell replication. One of the main abnormalitiesin human tumors cells is the loss of the GI phase checkpoint, which notonly permits cellular replication but also encourages genomicinstability. Consequently, enforcement of the G2/M checkpoint representsan attractive mode of action for new antineoplastic agents. G2/Mprogression is tightly regulated by several cellular macromolecules,including tubulins, and microtubule-associated proteins and motorproteins, such as kinesins and dynesins. An additional essentialregulator is the maturation/M-phase promoting factor comprisingCdk1/cyclin B. Cdkl/cyclin B itself is regulated by a complex group ofpositive and negative regulating kinases. In mammalian cells, theseinclude weel, mytt, cyclin activating kinase, Chk1 and cdsl kinases. hiaddition, Cdc25 phosphatases, which are also regulated by other kinasesand phosphatases, are responsible for the activation of Cdkl.

[0008] The ihibitors of tubulin polymerization or depolymerization arewidely available but only a few disrupters of other regulators of G2/Mprogression have been identified. For example, several small moleculeinhibitors of Cdc25 that block G2/M progression have been identified,but those compounds also affect GI transition (Tamura K, Rice RL, Wipf Pand Lazo JS (1999) Dual GI and G2/M phase inhibition by SC-uOa69, acombinatorially derived Cdc25 phosphatase inhibitor. Oncogene18:6989-6996, Tamura K, Southwick EC, Kerns J, Rosi K, Carr BI, Wilcox Cand Lazo JS (2000) Cdc25 inhibition and cell cycle arrest by a syntheticthioalkyl vitamin K analogue. Cancer Res 60:1317-1325). Others haverecently isolated a novel mitotic blocker that appears to act as aspecific inhibitor of a mitotic kinesin (Mayer TU, Kapoor TM, HaggartySJ, King RW, Schreiber SL and Mitchison TJ (1999) Small moleculeinhibitor of mitotic spindle bipolarity identified in a phenotype-basedscreen. Science 286:971-974). Nonetheless, there continues to be a greatneed for pharmacologically distinct agents both to investigate the G2/Mprogression process and as new pharmacophores for drug designstrategies.

[0009] The antifungal metabolites, preussomerins A-F, were identified in1990 by Gloer and coworkers during the course of an investigation ofchemical agents involved in interspecies competition among coprophilous(dung-colonizing) fungi. (Weber, H. A.; Baenziger, N. C.; Gloer, J. B. JAm. Chem. Soc. 1990, 112, 6718). In addition to those early reports fromPreussia isomera Cain samples, preussomerins were later also discoveredin the endophytic fungus Harmonerna dematioides (Polish J. D.;Dombrowski, A. W.; Tsou, N. N.; Salituro, G. M.; Curotto, J. E.Mycologia 1993, 85, 62). Other reports of an epoxy naphthalenediolspiroketal compound, bipendensin, have been published (Connolly, J. D.4th International Symposium and Pakistan-US. Binational Workshop onNatural Products Chemistry, Karachi, Pakistan, January 1990 andConnolly, J. D.: Structural elucidation of some natural products. InStudies in Natural Products Chemistry, Vol 9. Ed., Atta-ur-Rahman, pp.256-258, Elsevier Science Publishers B. V., Amsterdam, 1991).Bipendensin was isolated in very small amounts from wood samples of theAfrican tree Afzelia bipendensis. A compound having the same grossstructure as bipendensin was isolated in 1994 from an unidentifiedConiothyium fungus collected from forest soil on West Borneo, and wasnamed palmarumycin Cl, (Krohn, K.; Michel, A.; Florke, U.; Aust, H.-J.;Draeger, S.; Schulz, B. Liebigs Ann. Chem. 1994, 1099).

[0010] The pentacyclic palmarumycins are structurally unique naturalproducts with both antifungal and antibacterial activities, but theirantineoplastic effects are not well established. The naphthoquinoneacetals, palmarumycins, diepoxins and deoxypreussomerins arestructurally unique fungal metabolites with both antifungal andantibacterial activities, but their antiproliferative activity againstmalignant mammalian cells has not been extensively studied. (Wipf, P andJune JK (1998) Total synthesis of palmarumycin CPI and()-deoxypreussomerin A. J Org Chem 63:3530-3531: Schlingmann GRR, WestLP, Milne CJ and Carter GT (1993) Diepoxins, novel fungal metaboliteswith antibiotic activity. Tetrahedron Lett 34:7225-7228: Krohn K, MichelA, Florke U, Aust H-J, Draeger S and Schulz B (1994) Palmarumycins Cl-C16 from Coniothyrium: Isolation, structure elucidation, and biologicalactivity. Liebigs Ann Chem 1994:1099-1108). Biological studies have beenlimited due to the extraordinary synthetic challenges associated withthe extensive levels of oxygenation and the highly electrophilicfunctionality present in these spiroketal natural products.

[0011] Since its discovery in the early 1960s, thethioredoxin-thioredoxin reductase system has been the subject of intensepharmacological studies (Williams,C.H. Eur.JBiochem .2000, 267, 6101).The two redox active proteins have been isolated from many species, andtheir medical interest is based in part on their value as indicators ofwidespread diseases such as rheumatoid arthritis, AIDS, and cancer. Thecytosolic 12 kDa thioredoxin-1 (Trx-1) is the major cellular proteindisulfide reductase and its dithiol-disulfide active site cysteine pair(CXXC) serves as electron donor for enzymes such as ribonucleotidereductase, methionine sulfoxide reductase, and transcription factorsincluding NF-KB and the Ref-l -dependent AP-1 (Arn r,E.S.J.; Holmgren,A.Eur.JBiochem. 2000, 267, 6102). Therefore, thioredoxin-l is critical forcellular redox regulation, signaling, and regulation of protein functionas well as defense against oxidative stress and control of growth andapoptosis. (Davis,W.; Ronai,Z.; Tew,K.D. JPharm.Exp. Ther. 2001, 296,1). Thioredoxin-l acts in concert with the glutathione - glutathionereductase system but with a rate of reaction orders of magnitudefaster,and lack of cytosolic mammalian thioredoxin is embryonicallylethal. Eukaryotic thioredoxin reductases (TrxR) are 112-130 kDa,selenium-dependent dimeric flavoproteins that also reduce substratessuch as hydroperoxides or vitamin C (Williams, C.H.; Arscott, L.D.;Miller,S.; Lennon,B.W.; Ludwig,M.L.; Wang,P.-F.; Veine,D.M.; Becker,K.;Schirmer,R.H. Eur.JBiochem. 2000 , 267,6110). These reductases containredox-active selenylsulfide-selenolthiol active sites and are inhibitedby aurothioglucose and auranofin (K; 4 nM). (Becker,K.; Gromer,S.;Schirmer,R.H.; Mller,S. Eur.JBiochem.2000, 267, 6118). NADPH serves asreducing agent of Trx-l via TrxR.

[0012] Pathophysiological effects of Trx-l/TrxR are indicated by Trx-Ioverexpression in human tumors such as lung, colorectal and cervicalcancers and leukemia, and secreted Trx-1 stimulates cancer cell growthand decreases sensitivity to induced apoptosis (Powis,G.;Kirkpatrick,D.L.; Angulo,M.; Baker,A. Chem.-Biol.Interactions 1998 ,111,23). The Trx-l/TrxR system is therefore an important target forchemotherapeutic intervention. Alkyl 2-imidazolyl disulfides were foundto be inhibitors of Trx-1/TrxR with IC₅₀'s of 31/37 liM, respectively;these disulfides block MCF-7 human breast cancer cells in the G2/M phaseof the cell cycle and suppress the growth of several human primarytumors in the NCI 60 cancer cell line panel (Vogt,A.; Tamura,K.;Watson,S.; Lazo,J.S. JPharm.Exp.Ther. 2000 ,294 ,1070). A COMPAREanalysis revealed the most potent Trx-1/TrxR inhibitor known to date,the para -quinone NSC401005 which is the natural product pleurotin(Kunkel,M.W.; Kirkpatrick,D.L.; Johnson,J.I.; Powis,G. Anti-Cancer DrugDes. 1997, 12, 659). The IC 50 of NSC401005 was determined as 0.17 [tM;however, the average GI 50 of this compound in the 60 cell line panelwas only 21.5 atM. Although inhibitors of TrxR such as auranofin andnitrosoureas are quite effective, the search for new, more specific, andless toxic compounds is well justified. Therefore, a need exists in theart for new chemical compounds that block G2/M phase transition. Suchcompounds would find use as pharmacological probes and possible leadstructures for therapeutic agents. These compounds may includeinhibitors of the thioredoxin thioredoxin reductase system which areless toxic than current compounds.

SUMMARY OF THE INVENTION

[0013] The inventors have developed an efficient synthetic approachtoward palmarumycins, diepoxins and deoxypreussomerins and havegenerated a library of analogs. A number of these analogs inhibit thethioredoxin - thioredoxin reductase system. The inventors have examinedthe antiproliferative actions of pentacyclic palmarumycins against tumorcells using a temperature sensitive tsFT2 10 mouse mammary carcinomacell line and found that a new palmarumycin analog,[8-(furan-3-ylmethoxy)-l -oxo- 1 ,4-dihydronaphthalene-4-spiro-2′-naphtho[ 1“,8”-de] [1′,3′] [dioxin] herein termed SR-7, prominentlyblocked mammalian cell cycle transition in G2/M but not in G1 phase. Theinventors found no evidence for inhibition of the criticalmitosis-controlling cyclin-dependent kinase, Cdkl, or its regulator, thedual specificity phosphatase Cdc25. Moreover, Cdk1 washypophosphorylated and not directly inhibited by SR-7. SR-7 also failedin vitro to hypernucleate bovine tubulin, did not compete withcolchicine for tubulin binding, and only modestly blocked GTP-inducedassembly. In addition, SR-7 caused almost equal inhibition ofpaclitaxel-sensitive and -resistant cell growth. Moreover, unlikebenchmark tubulin disrupting agents, SR-7 did not causehyperphosphorylation of the antiapoptotic protein Bcl-2. Thus, SR-7 caninhibit G2/M transition by a mechanism that appears to be independent ofmarked tubulin disruption.

[0014] These and other advantages and benefits of the present inventionwill be apparent from the Detailed Description of the Invention hereinbelow.

BRIEF DESCRIPTION OF THE FIGS.

[0015] The present invention will now be described for the purpose ofillustration and not limitation in conjunction with the followingfigures wherein:

[0016]FIG. 1 shows the absolute stereochemistry of the preussomerins;

[0017]FIG. 2 depicts the acidic degradation of preussomerin A;

[0018]FIG. 3 illustrates preussomerins and deoxypreussomerins isolatedfrom a coelomycetes fungus;

[0019]FIG. 4 shows reaction of preussomerin G with strong nucleophile;

[0020]FIG. 5 depicts palmarumycins from Coniothyriumpalmarium;

[0021]FIG. 6 illustrates palmarumycins from an unidentified Coniothyriuspecies;

[0022]FIG. 7 illustrates treatment of palmarumycin C₉ and palmarumycinC₂ with methanolic HCl;

[0023]FIG. 8 shows cyclization of binaphthyl ether;

[0024]FIG. 9 depicts representative structurally related fungalmetabolites;

[0025]FIG. 10 shows a potential synthetic strategy toward palmarumycinCP1 and deoxypreussomerin A;

[0026]FIG. 11 illustrates preparation of 5-hydroxy-8-methoxy-1-tetralone;

[0027]FIG. 12 illustrates preparation of8-hydroxy-5-(8′-hydroxynaphthalene-l ′-yloxy)-1,2,3,4-tetrahydronaphthalene-1 -spiro-2“-dioxolane;

[0028]FIG. 13 shows oxidative cyclization of ketal 5;

[0029]FIG. 14 shows preparation of()-8-hydroxy-l-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[l “,8”-de] [1′,3 ′]dioxin;

[0030]FIG. 15 illustrates conversion of (I)-8-hydroxy-1-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[l“,8”-de] [1′,3′]dioxin topalmarumycin CPI;

[0031]FIG. 16 depicts an attempted epoxidation of palmarumycin CP,;

[0032]FIG. 17 depicts an attempted epoxidation of1-oxo-1,4,5,6,7,8-hexahydronaphthalene- 4-spiro-2′-naphtho[l “,8”-de][l′,3′]dioxin-8-spiro-2“′-dioxolane;

[0033]FIG. 18 illustrates treatment of ()-8-hydroxy-l-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[1“,8”-de][1′,3′]dioxin withhydrogen peroxide anion;

[0034]FIGS. 19A and B show generation of analogs from palmarnmycin CP₁;

[0035]FIG. 20 depicts treatment of palmarumycin CPI with 2-furylmethanol;

[0036]FIG. 21 illustrates deoxypreussomerin A and diepoxin analogs;

[0037]FIGS. 22A and B show the syntheses of TH- 169 and TH-223;

[0038]FIG. 23 illustrates cytotoxicity and G2/M phase inhibition bySR-7;

[0039]FIG. 24 illustrates cell growth inhibition of murine tsFT210cells;

[0040]FIG. 25 shows G1 transition in tsFT210 cells after treatment withSR-7;

[0041]FIG. 26 illustrates Cdkl dephosphorylation in the presence ofSR-7;

[0042]FIG. 27 depicts the ability of SR-7 to directly inhibit Cdklkinase activity;

[0043]FIG. 28 shows SR-7 effect on tubulin assembly; and

[0044]FIG. 29 depicts effects of SR-7 on Bcl-2 phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The following abbreviations are used herein: Cdk is cyclindependent kinase; DMSO is dimethyl sulfoxide; SC-uoa9 is 4-(benzyl-(2-[(2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylaminobutyric acid; and SDS- PAGE is sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The references cited in this detailed description ofthe present invention, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

[0046] The synthesis and initial antiproliferative evaluation of thepalmarumycin/diepoxin analogs have been described elsewhere (Wipf P,Jung J-K, Rodriguez S, Lazo JS Synthesis and biological evaluation ofDeoxypreussomerin A and Palmarumycin CP 1 and related naphthoquinonespiroketals. Tetrahedron 57:283-296 (2001)). The synthesis, biochemicaland cellular properties of the Cdc25 inhibitor, SC-aa69, have alsopreviously been published (Rice RL, Rusnak JM, Yokokawa F, Yokokawa S,Messner DJ, Boynton AL, Wipf P and Lazo JS (1997) A targeted library ofsmall molecule, tyrosine and dual specificity phosphatase inhibitorsderived from a rational core design and random side chain variation.Biochemistry 36:15965-15974), (Tamura K, Rice RL, Wipf P and Lazo JS(1999) Dual G1 and G2IM phase inhibition by SC-aa69, a combinatoriallyderived Cdc25 phosphatase inhibitor. Oncogene 18:6989-6996.). Curacin Awas prepared as described previously (Wipf P and Xu W (1996) Totalsynthesis of antimitotic marine natural product (+)- curacin A. J OrgChem 61:6556-6559.).

[0047] tsFT210 cells, which contain a temperature sensitive mutant formof Cdkl allowing for convenient cell cycle synchronization, were a giftfrom Dr. Chris Norbury (Oxford University, Oxford, UK) and weremaintained for no longer than 30 passages (Thing JP, Wright PS,Hamaguchi J, Lee MG, Norbury CJ, Nurse P and Bradbury EM (1990) TheFT21O cell line is a mouse G2 phase mutant with a temperature-sensitiveCDC2 gene product. Cell 63:313-324.). Paclitaxel-resistant (1 A9/PTX1 0,I A9/PTX22) and parental 1 A9 human ovarian carcinoma cells were giftsfrom Drs. Paraskevi Giannakakou and Tito Fojo of the National CancerInstitute (Bethesda, MD). The SV-40 large T antigen transformed cellshave been previously characterized (Vogt A, Wang AS, Johnson CS,Fabisiak JP, Wipf P and Lazo JS (2000) In vivo antitumor activity andinduction of insulin-like growth factor-l resistant apoptosis bySC-ctu,9. J Pharmacol Exptl Therap 292:530-537.). AntiCdkl (sc-54),antiCdc25, and antiBcl-2 (sc-509) antibodies were purchased from SantaCruz Biotechnology (Santa Cruz, CA). An agarose conjugate of antiCdklwas used for immunoprecipitation. Histone HI was obtained fromBoehringer Mannheim Co. (Indianapolis, IN) and [₇-³²P]ATP (1 OmCi/mmol)was from Amersham Life Science, Inc. (Arlington Heights, IL). [ring C,methoxy- ³H] colchicine (61.4 Ci/mmol, 2.3TBq/mmol) was from NEN(Boston, MA). Paclitaxel was obtained from the Drug Synthesis Branch ofthe National Cancer Institute. All other reagents were from Sigma Co.(St. Louis, MO) unless indicated otherwise.

[0048] The absolute stereochemistry of preussomerins has been assignedas shown in FIG. 1 on the basis of the isolation of known (-)-regioloneas a degradation product (Preussomerins A-F: Weber, H. A.; Gloer, J. B.J. Org. Chem. 1991, 56, 4355). Although the ketal linkages are resistantto acid hydrolysis at room temperature, vigorous cleavage conditions (6M HC1/ acetone, 1:1, 100 ° C, 12 h) yield (-)-regiolone as the majorproduct (Talapatra, S. L.; Karmacharya, B.; Shambhu, C. D.; Talapatra,B. Phytochemistry 1988, 27, 3929). Without being limited to any specifictheory, the inventors believe that conservation of the stereochemistryat the C- 1 ′position could be rationalized by a mechanism involvingprotonation at the C-2′position during the decomposition processfollowed by loss of the 9-OH proton and formation of an enol ether. Thisreaction is shown in FIG. 2. Hydrolysis of the remaining ketal linkagewould account for the formation of regiolone without loss ofstereochemical integrity at the hydroxylated benzylic carbon.

[0049] Even though preussomerin A has been reported to exhibit onlylow-micromolar cytotoxicity toward a mammalian cell line, (PreussomerinsA-F: Weber, H. A.; Gloer, J. B. J. Org. Chem. 1991, 56, 4355) a researchgroup reported that preussomerins and deoxypreussomerins showedpromising effects as ras famesyl-protein transferase (FTPase) inhibitors(Singh, S. B.; Zink, D. L.; Liesch, J. M.; Ball, R. G.; Goetz, M. A.;Bolessa, E. A.; Giacobbe, R. A.; Silverman, K. C.; Bills, G. F.; Pelaez,F.; Cascales, C.; Gibbs, J. B.; Lingham, R. B. J. Org. Chem. 1994, 59,6296).

[0050] Preussomerin G-I and deoxypreussomerin A and B, accompanied bypreussomerin D, were isolated from the fermentation broth of anunidentified coelomycetes fungus collected in Bajo Verde, Argentina.Structures of those compounds are shown in FIG. 3. IC₅₀'s of FTPaseinhibitory activities of preussomerins, deoxypreussomerins andderivatives of preussomerin G range between 1-20 ttM. Preussomerin G andpreussomerin D were the most active. Interestingly, deoxypreussomerins,which possibly are biosynthetic precursors of preussomerins had equal orbetter activities than preussomerins H and 1. Deoxypreussomerin A and Bwere also reported independently as antifungal agents and namedpalmarumycin C₂ and CP₂, respectively (Krohn, K.; Beckmann, K.; Florke,U.; Aust, H.-J.; Draeger, S.; Schulz, B.; Busemann, S.; Bringmann, G.Tetrahedron 1997, 53, 3101).

[0051] Preussomerin G can react with strong nucleophiles in a highlystereospecific Michael fashion to give a quantitative yield of theC-3′adduct as illustrated in FIG. 4. Without being limited to anyspecific theory, the inventors believe that steric hindrance may makethe top face of preussomerin G inaccessible to nucleophiles, and thusMichael addition can take place exclusively from the more accessiblea-face.

[0052] Additional naphthalenediol spiroketals of the palmarumycin familyhave been reported in the literature (Krohn, K.; Beckmann, K.; Florke,U.; Aust, H.-J.; Draeger, S.; Schulz, B.; Busemann, S.; Bringmann, G.Tetrahedron 1997, 53, 3101). Those metabolites produced by Coniothyriumpalmarium, are shown in FIG. 5 and those produced by an unidentifiedConiothyrium species are shown in FIG. 6.

[0053] Palmarumycin CP₃, CP₄, C₃, CIO and C[₂ show high antifungalactivity. It is theorized that the introduction of an oxygen functioninto the 8-position significantly increases the antifungal effect. Thechloroepoxide palmarumycin C₄ and palmarumycin Cg, isolated as anisomeric mixture of epoxides, completely inhibited germination andgrowth of garden cress. In most palmarumycins, only the relativeconfiguration was elucidated, except for palmarumycin CP4a and CP₅. Theabsolute configurations of the latter compounds were elucidated bycalculations. After computation of the circular dichroism (CD) spectraof six low energy conformers, Boltzmann-weighted addition and comparisonof the resulting averaged spectrum with the experimental data allowedthe assignment of the absolute configuration of palmarumycin CP₄a andCP₅ as shown in FIG. 5.

[0054] Krohn and coworkers proposed a biosynthesis of palmarumycin CPIbased on a 1,8- dihydroxynaphthalene or a suitable phenolic derivativeprecursor. (See also: Bode, H. B.; Wegner, B.; Zeeck, A. J. Antibiot.2000, 53, 153). According to their hypothesis, coupling could occur viaa phenol oxidation as often encountered in polyketide biosynthesis, andthe chlorinated palmarumycins could be derived from addition of chlorideions to epoxides. ( Herbert, R. B. The Biosynthesis ofSecondaryMetabolites, 2nd ed., Chapman and Hall, London, 1989 and O'Hagen, D. ThePolyketide Metabolites, Ellis Horwood, New York, 1991). To probe thismechanism, palmarumycin CP₂ and pahnarumycin C₉ were treated withmethanolic hydrochloric acid as shown in FIG. 7. As expected, formationof chlorinated palmarumycin C₄ from palmarumycin Cg could be detected byTLC. For the reaction of paimarumycin C₂, an intermediate chlorohydrinwas identified as the major isomer. This chlorohydrin slowly decomposedto palmarumycin Cl upon standing in chloroform solution. Palmarumycin C₂was recovered upon treatment with base. These experiments suggested tothe inventors a possible pathway to the chlorinated palmarumycins andhighlighted the unexpected stability of the naphthalenediol spiroketalwhich is not affected even by heating in acetic acid at 100 OC.

[0055] The open chain compound 1, shown in FIG. 8, has been isolatedfrom Coniothyrium palmarum. (Krohn, K.; Beckmann, K.; Aust, H.-J.;Draeger, S.; Schulz, B.; Busemann, S.; Bringmann, G. LiebigsAnn./Recueil 1997, 2531). This isolation offered the chance to probe thebiosynthetic hypothesis involving phenol oxidation. Upon exposure tosilver(II) oxide, the binaphthyl ether 1 cyclized to yield quinone ketal2 as depicted in FIG. 8. However, ketal 2 could not be detected in thefermentation broth of Coniothyrium palmarum. It is possible that a totalsynthesis of palmarumycins based on the phenolic oxidation of binaphthylethers could be achieved from compound 3, however, the inventors areunaware of further studies along these lines. Others have investigated abiomimetic cyclization approach with little success. (Ragot, J. P.;Alcaraz, M.- L.; Taylor, R. J. K. Tetrahedron Lett. 1998, 39, 4921).

[0056] Deoxypreussomerins and palmarumycins are structurally closelyrelated to the more recently isolated diepoxins (Schlingmann, G.; West,R. R.; Milne, L.; Pearce, C. J.; Carter, G. T. Tetrahedron Lett. 1993,34, 7225: Schlingrnann, G.; Matile, S.; Berova, N.; Nakanishi, K.;Carter, G. T. Tetrahedron 1996, 52, 435), to CJ-12,371 and CS-12,372(Sakemi, S.; Inagaki, T.; Kaneda, K.; Hirai, H.; Iwata, E.; Sakakibara,T.; Yamauchi, Y.; Norcia, M.; Wondrack, L. M. J. Antibiot. 1995, 48,134) and to spiroxins (McDonald, L. A.; Abbanat, D. R.; Barbieri, L. R.;Beman, V. S.; Discafani, C. M.; Greenstein, M.; Janota, K.; Korshalla,J. D.; Lassota, P.; Tischler, M.; Carter, G. T. Tetrahedron Lett. 1999,40, 2489). Some representative members are depicted in FIG. 9. (Forrelated compounds, see also: (a) Thiergardt, R.; Rihs, G.; Hug, P.;Peter, H. H. Tetrahedron 1995, 51, 733, (b) Chu, M.; Patel, M.; Pai,J.-K.; Das, P. R.; Puar, M. S. Bioorg. Med. Chem. Lett. 1996, 6, 579,(c)Chu, M.; Truumees, I.; Patel, M.; Das, P. R.; Puar, M. S. J. Aiztibiot.1995, 48, 329, (d) Chu, M.; Truumees, I.; Patel, M. G.; Gullo, V. P.;Pai, J.-K.; Das, P. R.; Puar, M. S. Bioorg. Med. Chem. Lett. 1994, 4,1539, (e) Chu, M.; Truumees, I.; Patel, M. G.; Gullo, V. P.; Puar, M.S.; McPhail, A. T. J Org. Chem. 1994, 59, 1222, and (f) Soman, A. G.;Gloer, J. B.; Koster, B.; Malloch, D. A Nat. Prod. 1999, 62, 659).

[0057] Antimicrobial, antifungal, and some anticancer activities havebeen identified for diepoxins and spiroxins. The inventors are aware ofa research group which isolated the novel fungal metabolites CJ-12,371and CJ-12,372 from a fermentation broth of an unidentified fungusN983-46. Those compounds showed DNA gyrase inhibitory activity. Thephospholipase D inhibitor Sch 53823 has the same gross structure aspalmarumycin CI , however, the melting point and optical rotation aredifferent, suggesting that palmarumycin Cl, and Sch 53823 arestereoisomers.

[0058] The combination of attractive biological activities and novelstructural features in the spirobisnaphthalene family of naturalproducts has attracted considerable interest from the synthetic organicconmmunity. In addition to the pioneering total syntheses ofpalmarumycin CPI and deoxypreussomerin (A, Wipf, P.; Jung, J.-K. J. Org.Chem. 1998, 63, 3530: Ragot, J. P.; Steeneck, C.; Alcaraz, M.-L.;Taylor, R. J. K. Perkin Trans. 1 1999, 1073 and Barrett, A. G. M.;Hamprecht, D.; Meyer, T. Chem. Commun. 1998, 809), innovative approachestoward diepoxin A, (Wipf, P.; Jung, J.-K. Angew. Chem. Int Ed. Engl.1997, 36, 764 and Wipf, P.; Jung, J.-K. J. Org. Chem. 1999, 64, 1092)preussomerins (G and 1, Chi, S.; Heathcock, C. H. Org. Lett. 1999, 1, 3)palmarumycin CP₂, palmarumycin C₁l, and CJ-12,371 have been reported inthe art since 1997.

[0059] The course of the inventors' work toward the total synthesis ofdiepoxin (Wipf, P.; Jung, J.-K. Formal Total Synthesis of (+) Diepoxin aJ. Org. Chem. 2000, 65, 6319) a potential synthetic strategy towardpalmarurycin CPI and deoxopreussomerin A was developed. This strategy issummarized in FIG. 10. Naphthalenediol spiroketal 4 was derived from abinaphthyl ether 5, and dehydrogenation at C(5) and C(6) in 4 should byfacilitated be the presence of the enone moiety. Compound 5 can easilybe prepared by an Ullmann ether coupling reaction with I -iodo-8-methoxynaphthalene 7. A modified experimental variant of the method ofGraybill et al., was used and the tetraline derivative (Graybill, B. M.;Shirley, D. A. J. Org. Chem. 1966, 31, 1221). Example 1

[0060] 5-hydroxy-8-methoxy-l-tetralone, compound 8 was prepared by amodified literature procedure (Newhall, W. F.; Harris, S. A.; Holly, F.W.; Johnston, E. L.; Richter, J. W.; Walton, E.; Wilson, A. N.; Folkers,K. J. Am. Chem. Soc. 1955, 77, 5646). Attempts for an Ullmann ethercoupling between 8 and 8-iodo-1-methoxynaphthalene 7 failed. Withoutbeing limited to any specific theory the inventors believe that thisfailure is quite likely due to the deactivating effect of the tetralonecarbonyl group. Coupling with ketal 6 is shown in FIG. I I was moresuccessful and resulted in a 78% yield of naphthyl ether 9 which wasfurther converted to ketone 10.

[0061] Although the inventors failed to demethylate ketal 9 with NaSEtin DMF or with BBr₃, demethylation of ketone 10 shown in FIG. 12, usingBBr₃ smoothly yielded compound 11 in 95% yield. The presence of a ketonefunction in 11 likely will retard the subsequent oxidative cyclizationwhich involves a very electron deficient transition state. Because theketone function in 11 was unreactive to acetalization conditions, thephenolic hydroxyl groups were first acetylated, and ketal 13 wassubsequently saponified to afford the oxidative cyclization precursor 5in good overall yield as shown in FIG. 12.

[0062] Oxidative cyclization of ketal 5 with Phl(OAc)₂ intrifluoroethanol afforded bisketal 14 in 75% yield is illustrated inFIG. 13. Unfortunately, deprotection of 14 under acidic conditions ledto complex mixtures.

[0063] Diol 11 was quantitatively reduced to triol 15, which wasoxidatively cyclized using Phl(OAc)₂ in trifluoroethanol to affordnaphthalenediol spiroketal 16 in 87% yield as depicted in FIG. 14.Further oxidation of the alcohol function of 16 was attempted with PCCand BaMnO₄ under buffered conditions, but failed to provide the desiredketone in acceptable yields. In contrast, when 16 was treated withactivated MnO₂ at room temperature, a clean conversion to the naturalproduct palmarumycin CPI was effected as shown in FIG. 15. For completeconversion of 16 to pf palmarumycin CPI, a large excess (more than 50equivalents) of MnO₂ was required, and a considerable amount of productremained adsorbed on MnO₂ and could not be recovered. When the reactionwas performed in dry benzene at reflux, the amount of MnO₂ required forthe complete conversion of 16 was decreased to -10 equivalents, but theresulting palmarumycin CPI was contaminated with a inseparablebyproduct. The inventors therefore used a two-step protocol in whichoxidation of 16 with Dess-Martin periodinane, purification of theintermediate ketone by column chromatography on SiO₂, and treatment with10 equivalents of MnO₂ in dry methylene chloride for 2 days at roomtemperature afford the target molecule in 60% yield. Palmarumycin CPIwas thus obtained in 35% overall yield in 8 steps from the knowntetralone 8.

[0064] Because of the close structural similarity between palmarumycinCPI and the farnesyl- protein transferase (FTPase) inhibitordeoxypreussomerin A, an epoxidation reaction of palmarumycin CPI wasattempted. However, treatment with hydrogen peroxide anion led todecomposition instead of epoxidation, and a mild epoxidizing agent,dimethyldioxirane also provided only decomposed products. Even afterprotection of the phenol function of palmarumycin CP, as the TBDMSether, no synthetically useful epoxidation could be achieved as shown inFIG. 16. Therefore, the inventors used earlier, more extensivelyprotected synthetic intermediates.

[0065] When compound 14 was treated with excess hydrogen peroxide anion,as shown in FIG. 17, monitoring of the reaction progress was difficultdue to the overlap of products with the starting material 14 on TLC. Thereaction mixture was thus quenched before complete consumption of 14. 'HNMR analysis of the crude product showed that mono- and diepoxides wereformed in a ratio of about 1 :1 with 1 0% remaining starting material.This result demonstrated that a regioselective epoxidation of thedisubstituted double bond of 14 in the presence of the internaltetrasubstituted double bond was unlikely to succeed.

[0066] In contrast, treatment of allylic alcohol 16 with hydrogenperoxide anion resulted in the is olation of the desired monoepoxide 20in 25% yield as depicted in FIG. 18. The relative configuration of theepoxide and the hydroxyl group was not determined. Peroxides and baseswere screened to optimize the epoxidation reaction. When cumenehydroperoxide and NaH were used at -20 OC, the epoxidation yieldincreased to 47%. The two step oxidation protocol developed for thesynthesis of palmarumycin CPI converted epoxy alcohol 20 to the desirednatural product in 55% yield. ()-Deoxypreussomerin A was synthesized in15% overall yield and 9 steps from the known 8. TABLE I PEROXIDE BASETEMPERATURE YIELD OF 20 Hydrogen peroxide K₂CO₃ rt 25% t-Butylhydroperoxide NaOH 0° C. 31% Cumene hydroperoxide NaOH 0° C. 40% Cumenehydroperoxide NaH 0° C. 45% Cumene hydroperoxide NaH −20° C.  47%

[0067] Synthesis of5-hydroxy-8-methoxy-1,2,3,4-tetrahydronaphthalene-1-spiro-2′-dioxolane(compound 6). To a solution of compound 8 (4.8 g, 25 nimol) and ethyleneglycol (3.1 g, 50 mmol) in benzene (700 mL) was added PPTS (0.3 g). Thereaction mixture was heated at reflux for 30 hours in a flask equippedwith a Dean-Stark apparatus, washed with 5% NaHCO₃ solution (2x 200 mL)and brine (300 mL), dried (Na₂SO₄), and concentrated in vacuo.Chromatography on SiO2 (hexanes/EtOAc, 2: 1) gave 5.32 g (90%) ofcompound 6 as a solid: Mp 139 - 140 ° C; IR (neat) 3359, 2928, 1583,1468, 1327, 1244, 1159, 1118, 1064, 1008, 945, 924, 864, 794, 716 cm⁴;'H NMR 6 6.55 (d, 1 H, J =8.8 Hz), 6.54 (d, 1 H, J =8.8 Hz), 5.42 (s, 1H, OH), 4.25 (t, 2 H, J =6.6 Hz), 4.07 (t, 2 H, J =6.6 Hz), 3.75 (s, 3H), 2.57 (t, 2 H, J =6.0 Hz), 1.93-1.80 (m, 4 H); ¹³C NMR 6 152.6,146.9, 128.3, 125.8, 115.2, 110.7, 108.1, 65.5, 56.6, 35.9, 24.0, 20.1;MS (El) m/z (rel intensity) 236 (M+, 94), 208 (100), 193 (19), 175 (11),164 (19), 149 (11), 134 (20), 121 (10), 106 (10), 99 (20), 77 (10), 65(9), 55 (13); HRMS (El) calcd for C₁₃HI₆0₄ 236.1049, found 236.1052.10067] Synthesis of8-methoxy-5-(8′-methoxynaphthalene-1′-yloxy)-3,4-dihydro-2H-naphthalen-1-one (compound 10). To a solution of compound 6 (4.72 g,0.02 mol) and 7 (8.52 g, 0.03 mol) in degassed pyridine (150 mL) wereadded K₂CO₃ (2.76 g, 0.02 mol) and Cu₂0 (286 mg, 0.002 mol). Thisreaction mixture was heated at reflux for 12 hours under a nitrogenatmosphere. After addition of additional Cu₂O (286 mg, 0.002 mol) to thesolution, heating was continued for 12 h. Pyridine was removed underreduced pressure and the residue was redissolved in EtOAc (300 nL). Itwas washed with water (100 mL) and brine (100 niL), dried (Na₂SO₄), andconcentrated in vacuo. Chromatography on SiO₂ (hexanes/EtOAc, 2:1) gave6.14 g (78%) of compound 9 as an oil. This oil was treated with TsOH(100 mg) in a mixture of acetone/water (7:1, 50 mL) for 7 hours at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was diluted with EtOAc (300 mL), washed with water (2x 100 mL)and brine (100 mL), dried (Na₂SO₄), and concentrated in vacuo.Chromatography on SiO₂ (hexanes/EtOAc, 1:1) gave 5.44 g (100%) ofcompound 10 as a colorless solid: Mp 152 - 153 ° C; IR(neat) 2952, 1696,1581, 1484, 1387, 1272, 1245, 1183, 1095, 980, 838, 821, 759 cm′; ′H NMR6 7.59 (dd, 1 H, J =8.2, 0.8 Hz), 7.45 (dd, 1 H, J =8.2, 1.0Hz), 7.37(q, 2 H, J =7.9Hz), 6.87 (dd, 1 H, J =7.6, 0.8 Hz), 6.81 (dd, 1 H, J=7.6, 0.8 Hz), 6.72 (d, 1 H, J=8.9 Hz), 6.67 (d, 1 H, J =9.1 Hz), 3.84(s, 3 H),3.74 (s, 3 H), 3.05 (t, 2 H, J=6.2 Hz), 2.67 (t, 2 H, J =6.3Hz), 2.11 (p, 2 H, J =6.4 Hz); 3 NMR 6 197.9, 156.2, 155.4, 152.7,149.3, 137.6,136.4, 126.7, 126.4, 124.1, 123.1, 121.7, 120.7, 118.8,115.9, 110.1, 106.1, 56.3, 56.0, 40.9, 24.1, 22.5; MS (El) m/z (relintensity) 348 (M+, 100), 319 (7), 305 (10), 291 (14), 261 (8), 218 (7),189 (12), 174 (24), 158 (45), 127 (34), 115 (29), 101 (10), 77 (15), 63(8); HRMS (El) Calcd for C₂₂H₂₀0₄ 348.1361, found 348.1361. .0068]Synthesis of 8-hydroxy-5-(8′-hydroxynaphthalen-1′-yloxy)-3,4-dihydro-2H-naphthalen-l-one (compound 11). To a solution of compound 10 (3.92 g,11.3 mmol) in CH₂C1₂ (120 rnL) was added a 1 M solution of BBr₃ inCH₂CI₂ (40 mL, 40 mmol) at -78 ° C. The reaction mixture was warmed toroom temperature, stirred for 12 h, poured into ice water (200 g) andextracted with CH₂CI₂ (2x 300 niL). The combined organic layers werewashed with brine (200 mL), dried (Na₂SO₄) and concentrated in vacuo.Chromatography on SiO₂ (hexanes/EtOAc, 8:1) gave 3.44 g (95%) ofcompound 11 as a colorless solid: Mp 165 - 166 ° C; IR (neat) 3403,2947, 1624, 1449, 1387,1343, 1289, 1213, 1167, 1024, 808, 749 cm¹; 'HNMR 6 12.46 (s, 1 H, OH), 9.02 (s, 1 H, OH), 7.50-7.32 (m, 4 H), 7.18(t, 1 H, J =8.0 Hz), 6.98 (dd, 1 H, J =7.2, 1.1 Hz), 6.93 (d, 1 H, J=8.9Hz), 6.40 (d, 1 H, J =7.7 Hz), 2.85 (t, 2 H, J =6.0 Hz), 2,70 (t, 2 H, J=6.3 Hz), 2.06 p, 2 H,J=6.4 Hz); ³C NMR6204.7, 161.1, 155.4,154.0,141.8, 137.2, 137.1, 131.1, 128.1, 125.5, 123.1, 119.3, 117.4, 117.1,114.9, 111.0, 107.4, 38.7,23.6,22.1;MS (El) m/z (rel intensity) 320 (M+,100), 287 (6), 263 (10), 247 (7), 177 (9), 159 (25), 144 (38), 131 (29),115 (34), 103 (15), 89 (10), 77 (23), 65 (14); HRMS (El) Calcd forC₂DHl₆0₄ 320.1049, found 320.1044.

[0068] Synthesis of acetic acid8-(4′-acetoxy-5′-oxo-5′,6′,7′,8′-tetrahydro-naphthalen-1′-yloxy)-naphthalen-l-yl ester (compound 12). To a solution of compound 11(487 mg, 1.52 mmol) in acetic anhydride (2 mL) was added sodium acetate(100 mg). The reaction mixture was heated to 95 ° C, stirred for 4 hoursand cooled to room temperature. The mixture was poured into ice water(100 g), stirred for 1 hours and extracted with ethyl acetate (100 mL).The ethyl acetate layer was washed with brine (50 mL), dried (Na₂SO₄)and concentrated in vacuo. Chromatography on SiO2 (hexanes/EtOAc, 2:1)gave 607 mg (99%) of compound 12 as an oil: IR (neat) 3059, 2951, 1765,1686, 1601, 1573, 1460, 1367, 1258, 1202, 1115, 1025, 898, 825, 760, 735cm ¹; ′H NMR 8 7.77 (d, 1 H, J =7.9 Hz), 7.58 (d, 1 H, J =8.0 Hz), 7.50(t, 1 H, J=8.0 Hz), 7.29 (t, 1 H, J=7.7 Hz), 7.18 (t, 1 H, J=7.4 Hz),7.16 (d, 1 H, J =7.6 Hz), 6.97 (d, 1 H, J=8.7Hz), 6.58 (dd, 1 H, J =7.7,0.7Hz), 2.91 (t, 2 H, J =5.7 Hz), 2.62 (t, 2 H, J =6.2Hz), 2.40 (s, 3H), 2.19 (s, 3 H), 2.07 (p, 2 H, J =6.4 Hz); ¹³CNMR 196.3, 170.3, 170.0,153.1, 150.8, 146.8, 146.0, 138.3, 137.1, 126.6, 126.4, 126.3, 126.2,125.8, 123.3, 123.1, 120.0, 119.4, 111.8, 40.1, 23.8, 22.0, 21.2, 21.1;MS (El) m/z (rel intensity) 404 (M+, 23), 362 (30), 320 (100), 202 (10),149 (21), 115 (12), 91 (33), 69 (18), 57 (28); HRMS (El) calcd forC₂4H₂₀0₆ 404.1260, found 404.1266.

[0069] Synthesis of8-hydroxy-5-(8′-hydroxynaphthalene-1′-yloxy)-1,2,3,4-tetrahydronaphthalene-l-spiro-2“-dioxolane (compound 13). To a solutionof compound 12 (240 mg, 0.593 mmol) and ethylene glycol (1.10 g, 17.79nunol) in benzene (20 mL) was added PPTS (75 mg, 0.297 mmol). Thereaction mixture was heated at reflux for 62 hours in a flask equippedwith a Dean-Stark apparatus, cooled to room temperature, diluted withbenzene (100 mL), washed with 5% NaHCO₃ solution (2x 50 mL) and brine(50 mL), dried (Na₂SO₄), and concentrated in vacuo. Chromatography onSiO₂ (hexanes/EtOAc, 1: 1) gave 205 mg (77%) of compound 13 as an oil.To a solution of compound 13 (175 mg, 0.39 mmol) in degassed THE/MeOH(15 mL, 2/1) was added lithium hydroxide monohydrate (41 mg, 0.98 mmol)at 0 ° C. The reaction mixture was stirred for 2 hours in an ice bath,neutralized with saturated ammonium chloride solution and extracted withethyl acetate (2x 100 mL). The combined organic layers were washed withbrine (100 mL), dried (Na₂SO₄), and concentrated in vacuo.Chromatography on SiO₂ (hexanes/EtOAc, 2: 1) gave 137 mg (96%) ofcompound 5 as a solid: Mp 174 - 175 ° C; IR (neat) 3405, 3318, 3057.2959, 2904, 1608, 1581, 1469,1402, 1365, 1301, 1253, 1220, 1182, 1157,1121, 1035, 944, 928, 878, 818, 759 cm-′; ′HNMR6 9.18 (s, 1 H, OH), 8.43(s, 1 H, OH), 7.46-7.34 (m, 3 H), 7.17 (t, 1 H, J =8.0Hz), 7.10 (d, 1 H,J=8.8 Hz), 6.96 (dd, 1 H, J=7.2, 1.1Hz), 6.85 (d, 1 H, J=8.8Hz), 6.45(d, I H, J =7.6Hz), 4.34-4.17 (m, 4 H), 2.68 (t, 2 H, J=6.3 Hz),1.99-1.95 (m, 2 H), 1.91-1.83 (mn, 2H); “³CNMR6 155.5, 154.8, 154.2,143.3, 137.0, 133.5, 127.8, 125.7, 124.4, 122.6, 120.6, 119.1, 116.1,114.9, 110.6, 109.8, 107.3, 63.9, 31.3, 23.5, 19.2; MS (El) m/z (relintensity) 364(M+, 100), 320 (55), 159 (11), 144 (24), 131 (14), 115(22), 77 (7), 55 (8); HRMS (El) calcd for C₂₂H₂₀0₅ 364.1311, found364.1311.

[0070] Synthesis ofI-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphthofl“,8“-

[0071] ′,3′]dioxin-8-spiro-2”′-dioxolane (compound 14). To a suspensionof compound 5 (58 mg, 0.159 mmol) in trifluoroethanol (20 mL) was addedPhl(OAc)₂ (62 mg, 0.191 mmol). The reaction mixture was stirred for 2hours at room temperature and NaHCO₃ (32 mg, 0.382 mmol) was added. Theresulting mixture was concentrated in vacuo and the residue was dilutedwith EtOAc (50 mL), washed with water (30 mL) and brine (30 mL), dried(Na₂SO₄), and concentrated in vacuo. Chromatography on SiO₂(hexanes/EtOAc, 4:1) gave 43 mg (75%) of compound 14 as an oily solid:IR(neat)3059,2949,2897, 1680, 1651, 1608, 1584, 1412, 1396, 1302, 1271,1144, 1096, 1052, 1031, 949, 825, 814, 757 cm-′; ′H NMR 6 7.52 (d, 2 H,J=8.1 Hz), 7.43 (t, 2 H, J =7.9 Hz), 6.93 (d, 2 H, J =7.1 Hz), 6.76 (d,1 H, J =10.3 Hz), 6.08 (d, 1 H, J =10.4 Hz), 4.41-4.36 (m, 2 H), 4.08-4.04 (m, 2 H), 2.75-2.65 (m, 2 H), 1.95-1.85 (m, 4 H); ¹³C NMR 6 182.4,154.1, 146.8, 136.4, 134.1, 134.0, 130.6, 127.6, 121.2, 112.9, 109.7,105.9, 92.8, 66.1, 35.6, 24.5, 19.5; MS (El) m/z (rel intensity) 362(M+, 100), 319 (39), 306 (16), 262 (15), 234 (9), 204 (10), 178 (16),131 (13), 115 (17), 99 (13), 84 (22), 55 (13); HRMS (El) calcd forC₂₂H,₈0₅ 362.1154, found 362.1160. .00721 Synthesis of()-8-hydroxy-1-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphthotl“8”-de][1′,3′]dioxin (compound 16). To a solution of compound11 (1.51 g, 4.72 nmmol) in Et₂O (70 nmL) was added in portions solidLiAlH4 (358 mg, 9.44 mmol) at 0 ° C. The solution was stirred for 2hours at 0 ° C, warmed to room temperature and stirred for an additional2 hours. The reaction mixture was carefully quenched with 5% sodiumbisulfate solution in an ice bath. After adding 40 mL of 5% sodiumbisulfate solution, the product was extracted with Et₂O (2x 150 mL). Thecombined ether layers were washed with brine (100 mL), dried (Na₂SO₄)and concentrated in vacuo. The resulting solid was added to drytrifluoroethanol (150 mL) and stirred until a fmie suspension wasobtained. After addition of Phl(OAc)₂ (1.67 g, 5.19 mmol), the mixturewas stirred for 30 min at room temperature, NaHCO₃ (1.0 g, 12 mmol) wasadded. The solution was concentrated in vacuo and the resulting residuewas diluted with EtOAc (300 mL), washed with water (100 mL) and brine(100 nmL), dried (Na2SO4), and concentrated in vacuo. Chromatography onSiO2 (hexanes/EtOAc, 2:1) gave 1.32 g (87%) of compound 16 as a yellowsolid: Mp 199 - 200 ° C; IR (neat) 3434, 2945, 1673, 1642,1630,1600,1409, 1374,1263, 1080, 944, 757 cmnf; ′HNMR 6 7.54 (d, 2 H, J =8.0 Hz),7.45 (td, 2 H, J =7.4, 2.2 Hz), 6.95 (td, 2 H, J =7.6, 0.7 Hz), 6.90 (d,1 H, J =10.4 Hz), 6.19 (d, 1 H, J =10.4 Hz), 4.82 (t, 1 H, J =4.9 Hz),3.31 (bs, 1 H, OH), 2.78-2.51 (m, 2 H), 1.98-1.90 (m, 3 H), 1.82-1.72(ni, 1 H); “³C NMR 8 185.8, 151.6, 146.8, 139.3, 135.6, 134.1, 129.1,127.7, 127.6, 121.3, 112.9, 109.8, 109.7, 92.6, 62.7, 29.6, 24.2, 17.7;MS (El) mn/z (rel intensity) 320 (M+, 100), 304 (30), 265 (35), 247(21), 235 (10), 219 (11), 197 (18), 169 (24), 160 (32), 144 (35), 133(35), 115 (50), 103 (16), 88 (13), 77 (28), 63 (17); HRMS (El) Calcd forC₂₀HI₆0₄ 320.1049, found 320.1039 10073] Synthesis of8-hydroxy-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphtho[1 “,81”- de][1′,3′] dioxin (palmarumycin CP,). To a solution of compound 16 (32 mg,0.1 mmol) in CH₂CI₂ (5 mL) was added Dess-Martin periodinane (64 mg,0.15 mmol) at room temperature. The reaction mixture was stirred for 2hours and diluted with EtOAc (30 mL). It was washed with 5% NaHCO₃solution (10 mL) and brine (15 mL), dried (Na₂SO₄), and concentrated invacuo. Chromatography on SiO₂ (hexanes/EtOAc, 2:1) gave 32 mg of ayellow residue which was treated with MnO₂ (Aldrich, 85% activated, 102mg, 1 mnmol, dried over P₂0₅ just before use) in dry CH₂C1₂ (5 mL) for 2days at room temperature. The reaction mixture was filtered throughcelite and washed with CH₂C1₂ (10 mnL). The combined solutions wereconcentrated in vacuo. Chromatography on SiO₂ (hexanes/EtOAc, 4: 1) gave19 mg (60%) of palmarumycin CPI as a yellow solid; Mp 170 ° C (dec.); IR(neat) 3053, 1659, 1602, 1449, 1409, 1372, 1341, 1269, 1237, 1110, 1073,942, 822, 746 cm¹l; ′HNMR 12.17 (s, 1 H, OH), 7.67 (t, 1 H,J =8.0Hz),7.58 (d, 2H, J =8.5Hz), 7.47 (t, 2 H, J=7.9 Hz), 7.46 (d, 1 H, J=7.8Hz), 7.14 (dd, 1 H, J=8.2, 1.1 Hz), 7.02 (d, 1 H, J=10.9 Hz), 6.98 (d, 2H, J=7.7 Hz), 6.37 (d, 1 H, J=10.9 Hz); ¹³C NMR 6 188.8, 161.9, 147.2,139.7, 138.8, 136.7, 134.2, 129.8, 127.7, 121.4, 119.7, 119.4, 113.8,113.0, 109.9, 92.9; MS (El) m/z (rel intensity) 316 (M+, 100), 288 (12),287 (19), 259 (8), 175 (11), 114 (45), 88 (11), 63 (9); HRMS (El) Calcdfor C₂₀HI₂0₄ 316.0736, found 316.0730. 10074] Synthesis of(+)-2,3-epoxy-8-hydroxy-1-oxo-1,2,3,4-tetrahydro-naphthalene-4-spiro-2′-naphtholl“,8”-de][1′,3′]dioxin [(+)-deoxypreussomerin A]. To a solutionof compound 16 (54.5 mg, 0.17 mmol) in THF (5 mL) was added cumenehydroperoxide (157 gL, 0.85 mmol) andNaH (60%, 6.5 mg, 0.17 mmol) at -20° C. The reaction mixture was stirred for 4 hours at -20 ° C, anddiluted with EtOAc (40 niL) and brine (5 mL). The separated organiclayer was washed with an additional brine (20 mI), dried (Na₂SO₄), andconcentrated in vacuo. Chromatography on SiO₂ (hexanes/EtOAc, 3:1) gave27 mg (47%/o) of monoepoxide 20. To a solution of this epoxide in CH₂CI₂(4 mL) was added Dess-Martin periodinane (51 mg, 0.12 mmol) at roomtemperature. The reaction mixture was stirred for 2 hours, diluted withEtOAc (30 mL), washed with 5% NaHCO₃ solution (10 niL) and brine (15mL), dried (Na₂SO₄), and concentrated in vacuo. Chromatography on SiO₂(hexanes/EtOAc, 2:1) gave 27 mg of a yellow residue which was treatedwith MnO₂ (Aldrich, 85% activated, 82 mg, 0.8 mmol, dried over P₂0₅ justbefore use) in dry CH₂CI₂ (5 mL) for 37 hours at room temperature. Themixture was filtered through celite and washed with CH₂CI₂ (10 mL). Thecombined solutions were concentrated in vacuo . Chromatography on SiO₂(hexanes/EtOAc, 3:1) gave 14.5 mg (26% from compound 16) of(+)-deoxypreussomerin A as a colorless solid: Mp 200 - 201 ° C; IR(neat) 3050, 1651, 1605, 1455, 1409, 1380, 1330, 1266, 1239, 1173, 1110,1061, 963, 920, 878, 820, 809, 759, 720 cm-′; ′HNMR 5 11.37 (s, 1 H,OH), 7.65 (t, 1 H, J =8.0 Hz), 7.60 (d, I H, J =8.6 Hz), 7.57 (d, 1 H, J=8.0 Hz), 7.53 (t, 1 H, J =8.3 Hz), 7.45 (t, 1 H, J =7.4Hz), 7.44 (d, 1H, J =7.9 Hz), 7.19 (dd, 1 H, J =7.6, 0.8 Hz), 7.14 (dd, 1 H, J =8.6,0.8 Hz), 6.92 (dd, 1 H, J =7.6, 0.7 Hz), 4.09 (d, 1 H, J =4.1 Hz), 3.68(d, 1 H, J =3.9 Hz); “³C NMR 8 196.6, 161.9, 146.9, 146.7, 137.8, 136.9,134.2,127.9,127.7, 121.5, 121.4, 120.1, 119.1, 112.8, 112.3,110.2,109.4,96.0,53.3; MS (El) m/z (rel intensity) 332 (M+, 100), 316 (28), 303(11), 287 (19), 173 (15), 145 (23), 132 (12), 114 (27), 89 (13), 74(14), 63 (12), 57 (7); HRMS(EI) Calcd for C₂₀HI20₅ 332.0685, found332.0688. 10075] Example 2 - Synthesis of(E)-8-(3-phenyl-allyloxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-de] [1′,3′] dioxin (compound 21). Generalprocedure for Mitsunobu reactions. A solution of palmarumycin CPI (7.4mg, 0.023 mmol), diphenylphosphino-polystyrene (82.1 mg, 1.41 mnmol/g,0.116 mmol) and cinnamyl alcohol (15.5 laL, 0.118 mmol) in dry CH₂CI₂(0.4 mL) was stirred for 30 min at room temperature and cooled to 0 ° C.Diethyl azodicarboxylate (DEAD) (18.0 [L, 0.114 mmol) was added to thereaction mixture at 0 ° C and stirring was continued for 24 hours atroom temperature. The reaction mixture was washed with 5% aqueous KOHsolution (0.5 mL), followed by 5% HCI (0.5 mL). The methylene chlorideextract was filtered, the resin was washed further with CH₂CI₂ (2x 0.5mL) and the solvent was concentrated. Chromatography on SiO₂(hexanes/EtOAc, 9:1) gave 1.8 mg (24%) of palmarumycin CPI and 5.0 mg(52%) of 21 as a colorless oil: ′H NMR o 7.70 (t, 1 H, J =8.0 Hz),7.60-7.56 (m, 3 H), 7.50-7.45 (m, 4 H), 7.37-7.20 (m, 4 ),6.99 (d, 2 H,J =7.3 Hz), 6.93 (bs, 1 H), 6.87 (d, 1 H, J =10.5 Hz), 6.49 (dt, 1 H, J=5.2, 16.0 Hz), 6.31 (d, 1 H, J =10.5 Hz), 4.93 (d, 2 H, J =5.2 Hz);HRMS(EI) Calcd for C₂₉H₂₀0₄ 432.1362, found 432.1362. 100761 Example 3 -Synthesis of (E)-8-(but-2-enyloxy)-l-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-de][1′,3′]dioxin (compound 22). According to the generalprocedure, palmarumycin CPI (2.4 mg, 0.008 mmol),diphenylphosphino-polystyrene (28.2 mg, 1.41 mmol/g, 0.040 mmol),2-buten-1-ol (3.3 p[L, 0.038 mnmol) and DEAD (6.0 liL, 0.038 mmol) indry CH₂CI₂ (0.2 mL) provided after 24 hours 2.5 mg (88%) of 22 as acolorless oil: ′H NMR o 7.68 (t, 1 H, J =8.0 Hz), 7.60-7.55 (m, 3 H),7.49 (d, 1 H, J =7.7 Hz), 7.46 (d, 1 H, J =8.2 Hz), 7.17 (d, 1 H, J =8.6Hz), 6.98 (d, 2 H, J =7.4 Hz), 6.85 (d, 1 H, J =10.5 Hz), 6.29 (d, 1 H,J =10.5 Hz), 6.03 (dq, 1 H, J =15.3, 6.5 Hz), 5.85-5.75 (m, 1 H), 4.69(d, 2 H, J =5.4 Hz), 1.80 (d, 3 H, J =6.2 Hz); HRMS(EI) Calcd forC₂4H,80₄ 370.1205, found 370.1214.

[0072] Example 4 - Synthesis of8-hexyloxy-1-oxo-1,4-dihydronaphthalene-4-spiro-2′- naphto[l“,8”-de][1′,3′ldioxin (compound 23). According to the general procedure,palmarumycin CPI (2.0 mg, 0.006 mmol), diphenylphosphino-polystyrene(31.3 mg, 1.41 mmol/g, 0.044 mmol), hexyl alcohol (4.0 tL, 0.031 mmol)and DEAD (5.0 nL, 0.032 mmol) in dry CH₂CI₂ (0.1 mL) provided after 43hours 1.3 mg (50%/o) of 23 as a colorless oil: ′H NMR 6 7.66 (t, 1 H, J=8.3 Hz), 7.57-7.50 (m, 3 H), 7.48-7.42 (m, 2 H), 7.14 (d, 1 H, J =8.3Hz), 6.96 (d, 2 H, J=7.5 Hz), 6.82 (d, 1 H,J=10.4Hz), 6.26 (d, 1 H, J=10.4Hz), 4.10 (t, 2 H, J=5.9 Hz), 2.30-1.20 (m, 11 H).

[0073] Example 5 - Synthesis of(E)-8-(hex-3-enyloxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-del[1′,3′ldioxin (compound 24, SR-4). According to thegeneral procedure, palmarumycin CPI (2.1 mg, 0.007 mmol),diphenylphosphino-polystyrene (23.9 mg, 1.41 mmol/g, 0.034 mmol),trans-3-hexen-1-ol (4.2 pLL, 0.034 mmol) and DEAD (5.2 tL, 0.033 nunol)in dry CH₂C1₂ (0.1 mL) provided after 67 hours 1.3 mg (43%) of 24 as acolorless oil: ′H NMR 6 7.68 (t, 1 H, J =8.3 Hz), 7.60-7.45 (m, 5 H),7.16 (d, 1 H, J =8.4 Hz), 6.97 (d, 2 H, J =7.5 Hz), 6.85 (d, 1 H, J=10.5 Hz), 6.28 (d, 1 H, J=10.4 Hz), 5.66-5.60 (m, 1 H), 5.45-5.30 (m, 1H), 4.15 (t, 2 H, J =6.9 Hz), 2.7-2.6 (m, 2 H), 2.4-2.3 (m, 2 H), 1.00(t, 3 H, J =6.4 Hz).

[0074] Example 6 - Synthesis of8-(3-methoxy-benzyloxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-de [1′,3′]dioxin (compound 25). A ccording to thegeneral procedure, palmarumycin CPI (2.0 mg, 0.006 mmol),diphenylphosphino-polystyrene (22.8 mg, 1.41 mmol/g, 0.032 mmol),3-mehoxybenzyl alcohol (4.0 [tL, 0.032 mmol) and DEAD (5.0 niL, 0.032mmol) in dry CH₂CI₂ (0.1 mL) provided after 45 hours 1.6 mg (67%) of 25as a colorless oil: ′H NMR 6 7.68- 7.54 (m, 4 H), 7.45 (t, 2 H, J =7.7Hz), 7.24-7.10 (m, 4 H), 6.97 (d, 2 H, J =7.5 Hz), 6.9-6.8 (m, 2 H),6.30 (d, 1 H, J =10.4 Hz), 5.29 (s, 2 H), 3.86 (s, 3 H).

[0075] Example 7 - Synthesis of8-(2-phenyl-ethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-del[1′,3′ldioxin (compound 26). According to the generalprocedure, palmarumycin CP, (2.0 mg, 0.006 mmol),diphenylphosphino-polystyrene (23.4 mg, 1.41 mmol/g, 0.033 nmmol),phenethyl alcohol (3.8 ,iL, 0.032 nmmol) and DEAD (5.0 ,uL, 0.032 mmol)in dry CH₂Cl₂ (0.2 mL) provided after 24 hours 1.0 mg (33%) of 26 as acolorless oil: ′H NMR 6 7.67-7.1 (m, 12 H), 6.98 (d, 2 H, J =7.5 Hz),6.86 (d, 1 H, J =10.5 Hz), 6.30 (d, 1 H, J =10.6Hz), 4.33 (t, 2 H, J=7.0 Hz), 3.27 (t, 2 H, J =7.0 Hz).

[0076] Example 8 - Synthesis of8-(furan-2-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-de][1′,31]dioxin (compound 27) (SR-7) and7-(furan-2-ylmethyl)-8-hydroxy-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto [1“,8”-de] [1′,3 ′] dioxin(compound 28). According to the general procedure, palmarumycin CPI (2.1mg, 0.007 mmol), diphenylphosphino- polystyrene (22.5 mg, 1.41 mmol/g,0.032 mmol), furfuryl alcohol (2.8 ,uL, 0.032 mmol) and DEAD (5.0 IL,0.032 mnmol) in dry CH₂CI₂ (0.2 mL) provided after 5 d 2.0 mg (71%) of27 and 1.0 mg (29%) of 28 as colorless oils. 27: ′H NMR 6 7.70-7.45 (m,6 H), 7.29-7.26 (m, 2 H), 7.05-6.95 (m, 2 H), 6.86 (d, 1 H, J =10.5 Hz),6.55 (bs, 1 H), 6.41 (bs, 1 H), 6.28 (d, 1 H, J=10.5 Hz), 5.23 (s, 2 H).28: ′H NMR 6 12.67 (s, 1 H), 7.61 (d, 2 H, J =8.2 Hz), 7.62-7.44 (m, 4H), 7.11 (d, 1 H, J =8.8 Hz), 7.00-6.92 (m, 3 H), 6.35 (d, 1 H, J =10.4Hz), 6.26 (t, 1 H, J =2.4 Hz), 5.91 (d, 1 H, J =3.2 Hz), 4.26 (s, 2 H).

[0077] Example 9 - Synthesis of(E,E)-8-(3,7-diemthyl-octa-2,6-dienyloxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphtol1“,8”-de[1l′,3′1dioxin (compound29). According to the general procedure, palmarumycin CPI (2.0 mg, 0.006mmol), diphenylphosphino-polystyrene (23.1 mg, 1.41 mmol/g, 0.033 mmol),geraniol (5.6 ,uL, 0.032 mmol) and DEAD (5.0 [tL, 0.032 mmol) in dryCH₂CI₂ (0.2 mL) provided after 29 hours 2.1 mg (83%) of 29 as acolorless oil: ′H NMR 6 7.67 (t, 1 H, J =8.2 Hz), 7.59-7.55 (m, 3 H),7.50-7.44 (m, 2 H), 7.16 (d, 1 H, J =8.2 Hz), 6.98 (d, 2 H, J =7.4Hz),6.85 (d, 1 H, J =10.5 Hz), 6.28 (d, 1 H, J =10.5 Hz), 5.56 (t, 1 H, J=6.0Hz), 5.10 (bs, 1 H), 4.79 (d, 2 H, J =6.2 Hz), 2.11 (bs, 4 H), 1.78(s, 3 H), 1.69 (s, 3 H), 1.62 (s, 3 H).

[0078] Example 10 - Synthesis of8-(furan-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-de][l′,3′jdioxin (compound 30, SR-10). Accordingto the general procedure, palmarumycin CPI (2.0 mg, 0.006 mmol),diphenylphosphino-polystyrene (23.4 mg, 1.41 mmol/g, 0.033 mmol),3-furamnethanol (2.8 ,uL, 0.032 mmol) and DEAD (5.0 [tL, 0.032 mmol) indry CH₂CI₂ (0.2 mnL) provided after 3 d 1.2 mg (50%) of 30 as acolorless oil: ′H NMR 6 7.63-7.45 (m, 5 H), 7.42-7.35 (m, 3 H), 7.16 (d,1 H, J=8.2 Hz), 6.89 (d, 2 H, J=7.4Hz), 6.77 (dd, 1 H, J=10.5, 1.3 Hz),6.51 (bs, 1 H), 6.20 (dd, 1 H, 3 =10.5, 1.3 Hz), 5.08 (s, 2 H); HRMS(EI)Calcd for C₂₅HI60₅ 396.0998, found 396.0997.

[0079] Example 11 - Synthesis of8-(pyridin-2-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-del[1′,3′ldioxin (compound 31). According to thegeneral procedure, palmarumycin CPI (2.2 mg, 0.007 mmol),diphenylphosphino-polystyrene (29.7 mg, 1.41 mmol/g, 0.042 rnmol),2-pyridylcarbinol (3.4 ttL, 0.035 rnmol) and DEAD (5.5 pL, 0.035 mmol)in dry CH₂CI₂ (0.2 mL) provided after 4 d 0.2 mg (9%) of palmarumycinCPI and 1.2 mg (43%) of 31 as a colorless oil: ′H NMR 6 8.50 (bs, 1 H),8.16 (bs, 1 H), 7.88 (bs, 1 H), 7.77-7.50 (m, 4 H), 7.51-7.45 (m, 3 H),7.4-7.3 (m, 1 H); 6.99 (d, 2 H, J =7.7 Hz), 6.92 (bd, 1 H, J =10.5 Hz),6.33 (d, 1 H, J =10.5 Hz), 5.42 (bs, 2 H); HRMS(EI) Calcd for C₂₆Hl₇NO₄407.1158, found 407.1139.

[0080] Example 12 - Synthesis of8-(pyridin-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[1“,8”-de][1′,3′]dio.xin (compound 32). According to thegeneral procedure, palmarumycin CPI (2.1 mg, 0.007 mmol),diphenylphosphino-polystyrene (23.8 mg, 1.41 numol/g, 0.034 nunol),3-pyridylcarbinol (3.3 gL, 0.033 numol) and DEAD (5.2 tL, 0.033 mmol) indry CH₂C1₂ (0.2 mL) provided after 5 d 0.3 mg (14%) of palmarumycin CP,and 0.9 mg (29%) of 32 as a colorless oil: ′H NMR 6 8.8-8.5 (m, 2 H),8.29 (d, 1 H, J =7.9 Hz), 7.74-7.40 (m, 8 H), 7.25-7.20 (m, 1 H), 6.97(d, 1 H, J =7.1 Hz), 6.88 (d, 1 H, J =10.5 Hz), 6.30 (d, 1 H, J =10.4Hz), 5.32 (s, 2 H); HRMS(EI) Calcd for C₂₆Hl₇NO4 407.1158, found407.1152.

[0081] Example 13 - Synthesis of8-(pyridin-4-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphtol1“,8”-de] [l′,3′]dioxin (compound 33). According to thegeneral procedure, palmarumycin CPI (2.0 mg, 0.006 mmol),diphenylphosphino-polystyrene (22.4 mg, 1.41 mmol/g, 0.032 mmol),4-pyridylcarbinol (7.6 mg, 0.069 mmol) and DEAD (5.0 pL, 0.032 mmol) indry CH₂Cl₂ (0.2 mL) provided after 7 d 0.6 mg (30%) of palmarumycin CPIand 0.5 mg (17%) of 33 as a colorless oil: ′H NMR 6 8.9-8.5 (m, 2 H),8.10 (bs, 1 H), 7.78-7.1 (m, 7 H), 7.00 (d, 2 H, J =7.4 Hz), 6.94 (d, 1H, J =10.5 Hz), 6.34 (d, 1 H, J =10.5 Hz), 5.40 (bs, I H), 4.80 (bs, 2H).

[0082] Example 14 - Synthesis of8-allyloxy-1-oxo-1,4-dihydronaphthalene-4-spiro-2′-naphto[l“,8”-de]f1′,3′1dioxin (compound 34). According to the generalprocedure, palmarumycin CPI (2.1 mg, 0.007 mmol),diphenylphosphino-polystyrene (23.5 mg, 1.41 rnmol/g, 0.033 mmol), allylalcohol (2.3 pL, 0.034 mmol) and DEAD (5.2 pL, 0.033 rnmol) in dryCH₂Cl₂ (0.2 mL) provided after 3 d 0.6 mg (33%) of palmarumycin CPI and1.8 mg (71%) of 34 as a colorless oil: ′H NMR 67.69 (t, 1 H, J =8.1 Hz),7.62-7.56 (m, 2 H), 7.49 (d, 1 H, J=7.5Hz), 7.46 (d, 1 H, J =8.3 Hz),7.17 (d, 1 H, J =8.3 Hz), 6.98 (d, 2 H, J=7.0Hz), 6.86 (d, 1 H, J =10.5Hz), 6.29 (d, 1 H, J =10.7 Hz), 6.20-6.06 (m, 1 H), 5.68 (dd, 1 H,J=17.2, 1.5 Hz), 5.38 (dd, 1 H, J =10.8, 1.5 Hz), 4.77- 4.74 (m, 2 H);HRMS(EI) Calcd for C₂₃HI₆0₄ 356.1049, found 356.1064.

[0083] Example 158-(4-methoxy-benzyloxy)-1-oxo-1,4-dihydronaphthalene4-spiro-2 -naphto [1,8 -de Ill ,3 ]dioxin (TH-39).A solution of palmarumycin CPI (20.1mg,0.0635 mmol), diphenylphosphino-polystyrene (230 mg, 1.41mmol/g,0.230 mmol) and 4-methoxybenzyl alcohol (39.6 pL,0.318 mmol) indry CH₂Cl₂ (2 mnL) was stirred for 45 min at room temperature and cooledto 0° C. Then DEAD (50 0 IL 0 318 mmol) was added dropwise to thereaction mixture to 0 ° C. . --: The solution was warmed to roomtemperature, stirred for 35 hours, diluted with additional CH₂C1₂, andwashed with 5% aqueous KOH solution followed by 5% HCl. The organicextracts were filtered. The resin was washed further with CH₂C1₂ and thecombined extracts were concentrated in vacuo. Chromatography on SiO2(Hexanes/EtOAc,25:1 I10:1 4:1)gave 6.1 mg (69%)of TH-39 I1 H NMR(CDC13)5 7.70-7.45 (m,8H),7.21 (dd,1 H, J=8.1,0.8 Hz),6.98 (t,4 H, J=8.2Hz),6.87 (d,1 H, J=10.5 Hz),6.31 (d,l H, J=10.5 Hz),5.26 (s,2 H),3.84(s,3 H);“³ C NMR (CDC1₃) 6 182.7, 159.2, 158.8, 147.4, 141.0, 135.1,134.7, 134.1, 132.2, 128.4, 128.3, 127.6, 121.2, 120.4, 115.9, 114.1,109.8, 93.4,70.7,55.3;HRMS (El) calcd for C₂gH₂₀Os 436.1311, found436.1323.

[0084] Example 16 TH-169 was prepared by hypervalent iodine oxidation asillustrated in FIG. 22A followed by transketalization with ethyleneglycol and 2-step aromatization. Spectroscopic data for TH-169.- Mp96.2-100.5 ° C;IR (neat)2956,2919,2852,1662,1617,1460,1393, 1344, 1296,1240, 1157, 1083,9 67, 843, 806, 746 cm -1 ; H NMR (CDC1₃)6 12.16 (s,lH),7.54 (t,l H, J=8.7 Hz),7.12 (d,1 H, J=7.6 Hz),7.01 (d,1 H, J=8.3Hz),6.85 (d,l H,J=l0.3 Hz),6.33 (d,l H, J=10.3 Hz),4.4-4.2 (m,4 H); “CNMR (CDCl₃) 6 189.6, 161.8, 144.1, 141.4, 136.2, 128.3, 118.9, 118.0,114.6, 99.9, 65.9; HRMS (El) calcd for C1₂H,00₄ 218.0579, found218.0571. 10090] Example 17 For the preparation of TH-223 ,the cesiumsalt of diol as illustrated in FIG. 22B was alkylated and cyclized underoxidative conditions. Spectroscopic data for TH-223: Mp 147.5-152.1 ° C;IR (neat) 2960, 2919, 2840, 1670, 1636, 1595,1475,1322,1258,1281,1094,1060 cm -1 ;1 H NMR (CDC1₃)6 7.69-7.55 (m,3 H),7.03(dd,l H ,J=7.8,1.5Hz),6.37 (d,l H, J=10.8 Hz),4.33 (td,2 H, J=12.6,2.5Hz),4.09 (dd,2 H, J=7.2,4.6 Hz),3.95 (s,3H),2.5-2.2 (m,l H),1.65-1.60(m,l H); 13 C NMR (CDC1₃)6 183.4, 159.4, 145.0, 134.6, 134.5, 130.9,119.3, 118.6, 112.5, 90.8, 61.3, 56.2, 25.1;HRMS (El) calcd for C₁₄HI₄0₄246.0892, found 246.0896. Biological Assays of Analogs

[0085] The successful development of efficient synthetic strategies forthe preparation of palmarumycin CPI and deoxypreussomerin A allowed theinventors to prepare analogs and investigate the biologicalstructure-activity relationship (SAR) of these compounds in more detail.The small library of palmarnmycin analogs was obtained by Mitsunobureaction of the natural product using polystyrene-boundtriphenylphosphine and is shown in FIG. 19. A total of 13 allylic andbenzylic alcohols were used for the coupling, and yields and ease ofpurification were greatly improved by the use of the polymer-boundreagent. In the treatment of palmarumycin CP, with 2- furyl methanol,the ether product 28 was accompanied by the C-alkylated phenol 27 asillustrated in FIG. 20. All other reactions produced a single isomer.The TH-39 through TH-140 series of analogs was prepared in a similarfashion in 10-70% yield from synthetic palmarumycin CP (FIG. 19B). Inaddition to these palmarumycin analogs, several diepoxin a derivativesas shown in FIG. 21 were also subjected to biological testing.

[0086] All moisture-sensitive reactions were performed under anatmosphere of N₂ or Ar and all glassware was dried in an oven at 140 ° Cprior to use. THF and Et₂O were dried by distillation over Na /benzophenone under a nitrogen atmosphere. Dry CH₂Cl₂ was obtained bydistillation from CaH₂. Dry DMF was obtained by distillation fromalumina under reduced pressure. Dry CF₃CH₂OH was obtained bydistillation from CaSO₄. Unless otherwise noted, solvents or reagentswere used without further purification. NMR spectra were recorded ateither 300 MHz / 75 MHz (′H / ¹³C NMR) or 500 MHz / 125 MHz (IH / ¹³CNMR) in CDCl₃ unless stated otherwise. Antiproliferative assay

[0087] Two widely used human breast cancer cell lines were evaluated forsensitivity to the cytotoxic effects of the naphthoquinone spiroketals.MCF-7 cells were originally derived from an adenocarcinoma of the breastand retain several characteristics of differentiated mamnmary epitheliumincluding the ability to process estradiol via cytoplasmic estrogenreceptors. MCF-7 cells express the tumor suppressor gene product p53,which is required for the programmed cell death or apoptosis caused bymany agents. (Foster, B. A.; Coffey, H. A.; Morin, M. J.; Rastinejad, F.Science 1999, 286, 2507).

[0088] MDA-MB-231 cells, which were also derived from an adenocarcinomaof the breast, are less differentiated than the MCF-7 cells and fail toexpress functional p53 or estrogen receptors. This class of tumor cellscontains important targets for new therapies, because loss of estrogenreceptor expression is associated with poor patient prognosis. (Osborne,C. K. Breast Cane. Res. Treatm. 1998, 51, 227).

[0089] All cells were tested for 72 hours with six concentrations ofcompounds ranging from 0.1 to 30 tiM to determine the concentrationrequired for 50% growth inhibition (IC₅₀). The inventors extrapolated todetermine the IC₅₀ for compounds with little cytotoxicity at 30 ,M, thehighest concentration tested. As can be appreciated by reference toTable II, 45% of the compounds (10/22) bad an IC₅₀ <3 ,M in both celltypes. Half of the compounds showed no selectivity to either human tumorcell type, while 32% of the compounds were more cytotoxic to MCF-7compared with MDA-MB-231 cells. This included compound 37, which was5-fold more cytotoxic to MCF-7 cells (IC₅₀ 4.6 vs. 23 ttM). The enhancedsensitivity of MCF-7 to these compounds may be due to the expression ofp53 in these cells. The assay used in these studies, however, did notspecifically measure apoptosis. At least one compound, 27, can arrestmammalian cells in the G2/M phase of the cell cycle. The five foldenhanced sensitivity of MDA-MB-231 cells to 24 compared to MCF-7 cellsis of interest, because the MDA-MB-23 1 cells lack both functionalestrogen receptors and p53. Thioredoxin reductase assay

[0090] Thioredoxin reductase was purified from human placenta aspreviously described (Oblong, J.E.; Gasdaska, P.Y.; Sherrill, K.;Powis,G. Biochemistry 1993, 32, 7271) and recombinant human Trx-l wasprepared as previously described (Gasdaska, P.Y. ; Oblong, J.E.;Cotgreave, I.A.; Powis,G. Biochem.Biophys.Acta 1994 ,1218 ,292). TrxRand Trx-l/TrxR activities were measured spectrophotometrically usingpreviously published microtiter plate colorimetric assays, based on theincrease in absorbance at 405 nm which occurs as dithionitrobenzoic acid(DTNB) is reduced by the enzyme-mediated transfer of reducingequivalents from NADPH (Gasdaska et al., Biochem.Biophys.Acta 1994 ,1218,292). Trx-l/TrxR-dependent insulin reducing activity was measured in anincubation with a final volume of 60 tL containing 100 mM HEPES buffer,pH 7.2,5 mM EDTA (HE buffer),l rmM NADPH,l.0 FM TrxR,0.8 ItM Trx-l and2.5 mg/mL bovine insulin. Incubations were for 30 min at 37 ° C inflat-bottom 96-well microtiter plates. The reaction was stopped by theaddition of 100 FL of 6 M guanidine-HCl, 50 mM Tris, pH 8.0, and I0 mMDTNB ,and the absorbance measured to 405 nm. TrxR activity was measuredin a final incubation volume of 60 tL containing HE buffer, 10 mM DTNB,1.0 FM TrxR and 1 mM NADPH. Compounds were diluted in HE buffer andadded to the wells as 20 IL aliquots, and TrxR was then added,also as 20FtL aliquots in HE buffer. To start the reaction NADPH and DTNB wereadded as a 20 RL aliquot in HE buffer and the plate was moved to theplate reader which had been preheated to 37 ° C. The optical density at405 nm was measured every 10 s and initial reaction rates were measured.

[0091] Table II summarizes Trx-l/TrxR assay data as well as growthinhibition values for selected compounds. The most active compoundsinhibited Trx-I/TrxR with IC50 values from 0.35 to low micromolar. Inparticular, palmarumycin CPI rivaled the most active known inhibitor ofthe thioredoxin system, pleurotin, in activity. Palmarumycin CPI alsodemonstrated considerable (>30 fold) selectivity for Trx-l over TrxR.Alkylation at the phenol as shown in the SR-series of analogs mostlyabolished activity, with the exception of SR-10 ,a 3-furylmethylderivative, and SR-14 ,an allylated phenol which were nonetheless >50fold less active. For the most part, this trend is continued in theTH-series, but several derivatives show more significant affmity to thethioredoxin thioredoxin reductase system. Specifically,TH-40,TH-44,andTH-62 have IC₅o values from 4.8 to 13.4 I. The former two are closelyrelated to SR-1 4 ,but the activity of TH-62 is unexpected given thelack of activity of the closely related TH-63 -66 . The beneficialeffects of the free phenol group in the palmarumycin pharmacophore forTrx-l/TrxR inhibition are most strikingly demonstrated in the comparisonof TH-169 and TH-223. Only the free phenol TH-169 maintains significantactivity while the methyl ether TH-223 is practically inactive. Thecomparison between palmarumycin CP, and TH-1 69 also demonstrates thecontribution to activity by the naphthalenediolketal; a replacement withthe 1,3-dioxolane group decreases activity approximately 10 fold and,most significantly, reduces the Trx-1 selectivity from >30 toapproximately 2 fold. TABLE II IC₅₀ values [μM] and 50% cell growthinhibition activities [μM] of Trx-1/TrxR inhibitors. No. Compound TrxRTrx-1/TrxR MDA-MB-231 MCF-7 palmarumycin 12.0 0.35 2.4 1 CP 1 diepoxin13.5 4.5 2 1.5 21 SR-1 nd >50 7.5 7.9 22 SR-2 nd >50 2.9 1.3 23 SR-3nd >50 13.6 13.4 24 SR-4 nd >50 9.2 >30 25 SR-5 nd >50 2.7 2.3 26 SR-6nd >50 4.6 3.9 27 SR-7 nd >50 2.5 1.1 29 SR-9 nd >50 2 4.6 30 SR-10 >5023.2 2 2 31 SR-11 >50 41.8 2.8 2 32 SR-12 >50 >50 1.4 1.5 33SR-13 >50 >50 7.3 8 34 SR-14 >50 23.2 2.7 2 TH-39 >50 >50 2.2 0.7TH-40 >50 4.8 8.2 7.8 TH-44 >50 13.4 4.7 4.3 TH-48 >50 >50 9.3 >10TH-49 >50 >50 8 >10 TH-62 20.1 10.2 7.8 5.7 TH-63 >50 >50 >10 >10TH-64 >50 >50 >10 >10 TH-65 >50 >50 4.9 5 TH-66 >50 42.4 5.2 5.5 TH-126nd >50 3.6 2 TH-139 nd >50 5.3 4.3 TH-140 nd >50 4.5 1.9 TH-169 8.8 3.44.3 4.2 TH-223 >50 40.2 5 4.4

[0092] Two compounds, 24 referred to herein as SR-4 and 27 referred toherein as SR-7, were examined in greater detail as to their effects onbiological activity. The proliferation of MCF-7, MDA-MB-231 and SV40transformed mouse embryonic fibroblasts was measured by a previouslydescribed colorimetric assay (Wipf P, Jung J-K, Rodriguez S, Lazo JSSynthesis and biological evaluation of Deoxypreussomerin A andPalmarumycin CP 1 and related naphthoquinone spiroketals. Tetrahedron57:283-296, 2001 and Vogt A, Rice RL, Settineri CE, Yokokawa F, YokokawaS, Wipf P and Lazo JS (1998) Disruption of insulin-like growth factor-Isignaling and down-regulation of Cdc2 by SC-oo:69, a novel smallmolecule antisignaling agent identified in a targeted array library. JPharmacol Exptl Therap 287:806-813.). Briefly, the inventors seeded4,000- 6,000 cells per well in microtiter plates. Cells were allowed toattach overnight and treated with vehicle or compounds for 72 h, afterwhich the medium was replaced with serum free medium containing 0.1%3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide. Plateswere incubated for 3 hours in the dark and total cell number wasdetermined by spectrophotometrically at 540 nm as previously described(Vogt A, Rice RL, Settineri CE, Yokokawa F, Yokokawa S, Wipf P and LazoJS (1998) Disruption of insulin-like growth factor-i signaling anddown-regulation of Cdc2 by SC-(xa69, a novel small moleculeantisignaling agent identified in a targeted array library. J PharmacolExptl Therap 287:806-813.). The growth inhibition of IA9, 1A9/PTX1O and1 A9/PTX22 cells was also measured with a slightly differentcolorimetric assay. Cells were maintained in RPMI 1640 medium containing10% fetal bovine serum and the paclitaxel-resistant cells also contained17 nM paclitaxel and 10 tiM verapamil. Cells were plated (2,000/well) in96- well plates and allowed to attach and grow for 72 hours (paclitaxeland verapamil were removed from resistant cell medium two weeks prior tothis plating). They were then treated with 0.5% DMSO vehicle control or0.08-10 lM compound. The number of cells was determinedspectrophotometrically at 490 nm minus absorbance at 630 nm afterexposure to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumand N- methyldibenzopyurazine methyl sulfate.

[0093] Exponentially growing MCF-7 (FIG. 23, Panel A) or MDA-MB-231(Panel B) cells were exposed to various concentrations of palmarumycinCPI, diepoxin (7, SR-7 or SR-4 for 72 hours and the cell numberdetermined using 0.1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as described elsewhere herein. Control values werevehicle treated cells. N=8, Bars=SEM. MCF-7 cells showed similarsensitivity to the antiproliferative actions of palmarumycin CPI,diepoxin a and SR-7 with an IC₅₀ of approximately 1-2 iM (Panel A). Incontrast, the close structural analog SR-4 had considerably lessantiproliferative activity against these cells with an IC₅₀ greater than10 liM. Because of the potential importance of the tumor suppressor genep53 and the estrogen receptor in controlling the cellular response tocytotoxic agents, the inventors also examined the sensitivity ofMDA-MB-231 cells, which lack functional p53 and estrogen receptors. Ascan be appreciated by reference to FIG. 23, Panel B, MDA-MB-231 cellswere equally sensitive to palmarumycin CPI, diepoxin a and SR-7. Whencompared to SR-7, however, SR-4 was again less active revealing theimportance of the 2-furyl moiety in the C8 position of SR-7. As might beexpected, these p53 and estrogen receptor deficient cells were generallyless sensitive to both natural products and analogs when compared toMCF-7 cells. The differential cytotoxicity of the SR-7:SR-4 pair wasconfirmed when the inventors tested mouse embryonic fibroblaststransformed with SV40 large T antigen; the inventors found 3 PM SR-7,palmarumycin CPI, and diepoxin a were required to inhibit growth by 50%,while no significant inhibition was seen with 10 IAM SR-4, the highestconcentration tested (data not shown). Flow cytometry analysis

[0094] tsFT210 cells were seeded at 2 x 10⁵ cells/ml and maintained at32.0° C as previously described (Tamura K, Rice RL, Wipf P and Lazo JS(1999) Dual GI and G2/M phase inhibition by SC-au69, a combinatoriallyderived Cdc25 phosphatase inhibitor. Oncogene 18:6989-6996, Th′ng JP,Wright PS, Hamaguchi J, Lee MG, Norbury CJ, Nurse P and Bradbury EM(1990) The FT210 cell line is a mouse G2 phase mutant with atemperature-sensitive CDC2 gene product. Cell 63:313- 324.). Cellproliferation was blocked at G2 phase by incubation at 39.4° C for 17 h.The synchronized cells were then released by re-incubating at 32.0° Cand treated immediately with 0-1 0 . M SR-7, 1 MM nocodazole or 100 EMSC-ca.69, respectively, to probe for G2/M arrest. Cells were treated 6hours after G2/M release to determine GI arrest. The inventors used 100gM SC- (ox69 and 50 JiM roscovitine as positive control compounds for GIarrest. A final concentration of 0.5% DMSO was used for all compoundsand as a negative control. For both G2/M and GI blockage studies,treated cells were incubated at 32.0° C for an additional 6 hours aftereach drug exposure, and then harvested with phosphate buffered saline at5 x I0⁵ cells/ml. The harvested cells were stained with a solutioncontaining 50 lug/ml propidium iodide and 250 ptg/ml RNase A. Flowcytometry analysis was conducted with a Becton Dickinson FACS Star(Franklin Lakes, NJ).

[0095] When incubated at the permissive temperature of 32.0° C, tsFT210cells had a normal cell cycle distribution (FIG. 24, Panel A); whenincubated at the non-permissive temperature of 39.4° C for 17 h, cellsarrested at G2/M phase (4C), due to Cdkl inactivation (Panel B). WhenG2/M arrested cells were cultured at the permissive temperature for 6hours with DMSO vehicle alone, the inventors saw clear evidence of entryinto Gi (2C) (Panel C), although a small fraction of cells remained inthe G2/M phase. This G2/M retention at 4C is probably due to theextended cell cycle blockage at 39.4° C (Osada H, Cui CB, Onose R andHanaoka F (1997) Screening of cell cycle inhibitors from microbialmetabolites by a bioassay using a mouse cdc2 mutant cell line, tsFT210.Bioorg Med Chem 5:193-203). Treatment with 1 liM nocodazole blocked cellpassage through G2/M (Panel D).

[0096] To determine the effect of SR-7 on G2/M cell cycle transition,the inventors treated cells with 2.5 to 10 tM SR-7 for 6 hours afterreleasing cells at 32.0° C. As indicated in Panels E-H, SR- 7 caused aconcentration-dependent arrest in the G2/M phase, with obvious blockageeven with 2.5 liM SR-7. The G2/M inhibition was similar to that seenwith the previously reported and structurally unrelated compound SC-caa9(Panel 1). SC-aa69 is an inhibitor of the Cdc25 family of phosphatasesthat controls cell cycle checkpoints (Rice RL, Rusnak JM, Yokokawa F,Yokokawa S, Messner DJ, Boynton AL, Wipf P and Lazo JS (1997) A targetedlibrary of small molecule, tyrosine and dual specificity phosphataseinhibitors derived from a rational core design and random side chainvariation. Biochemistry 36:15965-15974).

[0097] The inventors examined Gl transition in tsFT210 cells after SR-7treatment. tsFT210 cells were cultured at the permissive temperature of32.0° C (FIG. 25, Panel A) and then incubated for 17 hours at thenon-permissive temperature of 39.4° C (Panel B). Cells were releasedfrom the G2/M block by incubation at 32.0° C for 6 hours (Panel C), andthen incubated for an additional 6 hours in the presence of variousagents. These were: DMSO vehicle (Panel D), 50 ,uM roscovitine (PanelE), 5 pM SR-7 (Panel F), 10 liM SR-7 (Panel G), or 100 tM SC-aa69 (PanelH). Fluorescence corresponding to 2C and 4C DNA contents is representedby vertical bars. These results were replicated in a second independentexperiment. Cells that were treated with the DMSO vehicle passed throughG1 phase as expected and produced the predicted broad S phase peakbetween diploid (2C) and tetraploid (4C) states (D), while cells exposedcontinuously to 50 ,uM roscovitine were blocked and did not pass throughG1 (Panel E). As illustrated in Panels F and G, cells treated with 5 or10 tM SR-7 were not delayed at G1. As expected from previous studies(Tamura K, Southwick EC, Kerns J, Rosi K, Carr BI, Wilcox C and Lazo JS(2000) Cdc25 inhibition and cell cycle arrest by a synthetic thioalkylvitamin K analogue. Cancer Res 60:1317-1325.), the dual phase-specificinhibitor SC-aa89 caused a prominent Gi block and also prevented cellsthat were at the G2/M interphase from progressing, which resulted in twoprominent cell cycle peaks (Panel H). Western blotting and Cdkl assays.

[0098] tsFT210 cells were harvested using the same procedure for cellsynchronizing and drug exposure as described above for the G2/M flowcytometric analysis. The protein lysates were analyzed by Westernblotting for Cdkl as described previously (Tamura K, Southwick EC, KernsJ, Rosi K, Carr BI, Wilcox C and Lazo JS (2000) Cdc25 inhibition andcell cycle arrest by a synthetic thioalkyl vitamin K analogue. CancerRes 60:1317-1325.). The inventors used Cdkl isolated from human MCF-7cells to assay for in vitro inhibition of enzyme activity because ofavailable antibodies and convenience. Asynchronous cells grown at 37° Cin 5% CO₂ in DMEM containing 10% fetal bovine serum were treated withlysis buffer and harvested as previously described (Vogt A, Wang AS,Johnson CS, Fabisiak JP, Wipf P and Lazo JS (2000) In vivo antitumoractivity and induction of insulin-like growth factor-I resistantapoptosis by SC-uox9. J Pharmacol Exptl Therap 292:530-537.). Cdklkinase activity assay was performed as previously described (Yu L,Orlandi L, Wang P, Orr MS, Senderowicz AM, Sausville EA, Silvestrini R,Watanabe N, Piwnica-Worms H, O° Connor PM (1998) UCN-0 1 arogates G2arrest through a Cdc2-dependent pathway that is associated withinactivation of the WeelHu kinase and activation of the Cdc25Cphosphatase. J Biol Chem 273: 33455-33464.). Briefly, 2 mg of theprotein lysates were incubated with antiCdkl antibody agarose conjugatefor 2 hours at 4 ° C. The immunoprecipitates were treated in vitro withDMSO vehicle, 300 nM flavopiridol or 10 iM SR-7 for 20 min at 30° C. Thetreated immunoprecipitates were re-incubated in 20 ptl of kinasereaction buffer (Yu L, Orlandi L, Wang P, Orr MS, Senderowicz AM,Sausville EA, Silvestrini R, Watanabe N, Piwnica-Worms H, O° Connor PM(1998) UCN-01 arogates G2 arrest through a Cdc2-dependent pathway thatis associated with inactivation of the WeelHu kinase and activation ofthe Cdc25C phosphatase. J Biol Chem 273: 33455-33464.) for an additional20 min at 30° C with 3 pg of histone HI, 20 mM Tris-HCl, 10 mM MgCl₂, 5[tM cold ATP and 10 uCi of [-³²P]ATP. Histone HI was separated fromother proteins by SDS-PAGE and analyzed for incorporation of radioactivephosphate with a Molecular Dynamics STORM 860 Phospholmager (Sunnyvale,CA).

[0099] The major controlling molecule for G2/M transition is the cyclindependent kinase, Cdkl, whose cellular activity is tightly regulated byphosphorylation (Pines J (1999) Four-dimensional control of the cellcycle. Nature Cell Biol 1:E73-79., Hunter T and Pines J (1994) Cyclinsand cancer II: cyclin D and CDK inhibitors come of age. Cell79:573-582). Therefore, the inventors performed Western blotting ontsFT210 cell extracts to determine the Cdkl phosphorylation level in thepresence or absence of SR-7. G2/M synchronous tsFT210 cells were treatedwith vehicle or various compounds and permitted to re-enter the cellcycle by culturing at 32.0° C. The inventors isolated protein lysatesfrom cells that were not incubated (0 h) or from cells incubated for 2-6hours at the permissive temperature in the presence of a compound orvehicle. The protein lysates were analyzed by Western blotting for Cdklcontent and phosphorylation status as described elsewhere herein.

[0100]FIG. 26 depicts the results of the Cdkl assay in which DMSOcontrol, 0-6 hours is shown in lanes 1-4. Nocodazole, 1 VM for 6 hoursin lane 5, SR-7, 10 and 20 ,uM for 6 hours in lanes 6 and 7 and,SC-ao(69, 50 ,IM for 6 hours in lane 8. These results were confirmed ina second independent experiment. Similar to previous observations,approximately 50% of Cdkl was in the mitotic-inactivehyperphosphorylated form as reflected by a slower migrating Cdkl (lane1). The phosphorylation of Cdkl decreased gradually after cells werereleased from G2/M block, and most of the Cdkl was dephosphorylated and,thus activated, 6 hours after G2/M release, even in the presence of theDMSO vehicle (lanes 2-4). When the inventors incubated cells with 1 FMnocodazole, which caused a G2/M arrest, no hyperphosphorylation of Cdklwas seen, consistent with its proposed inhibitory activity after Cdklactivation (lane 5). Similarly, Cdkl was completely dephosphorylated inthe presence of either 10 or 20 gM SR-7 (lanes 6 and 7). In contrast,treatment with 100 IM SC-a cc9, which also causes G2/M block (Tamura K,Rice RL, Wipf P and Lazo JS (1999) Dual GI and G2/M phase inhibition bySC-aoa69, a combinatorially derived Cdc25 phosphatase inhibitor.Oncogene 18:6989-6996), yielded a hyperphosphorylated Cdkl (lane 8). Invitro studies confirmed that SR-7 and SR-4 at 30 liM caused noinhibition of recombinant Cdc25, VHR or PTPIB activity; even at 100 MMthe inventors found #16% inhibition (data not shown). -01071 Theinventors also examined the ability of SR-7 to directly inhibit Cdklkinase activity. Lysates from asynchronous MCF-7 cells wereimmunoprecipitated with anti Cdkl antibody coupled to agarose and theresulting immunoprecipitate treated with DMSO vehicle, 300 nMflavopiridol or 30 pM SR-7 for 20 min. The resulting immunocomplexeswere tested for their ability to phosphorylate histone HI using[y-³²P]ATP. FIG. 27, Panel A shows the phosphorylation of histone Hi.Panel B shows the total Cdkl protein level as measured with an antiCdklantibody. Panel C shows the quantification of the intensity of histoneHI phosphorylation normalized to the total Cdkl amount. Tubulinpolymerization

[0101] Tubulin without microtubule-associated proteins was isolated fromfresh bovine brains (Hamel E and Lin CM (1984). Separation of activetubulin and microtubule-associated proteins by ultracentrifugation andisolation of a component causing the formation of microtubule bundles.Biochemistry 23:4173-4184). Inhibition of assembly reactions was carriedout as described previously (Verdier-Pinard P, Lai J-Y, Yoo H-D, Yu J,Marquez B, Nagle DG, Nambu M, White JD, Falck JR, Gerwick WH, Day BW andHamel E (1998) Structure-activity analysis of the interaction of curacinA, the potent colchicine site antimitotic agent, with tubulin andeffects of analogs on the growth of MCF-7 breast cancer cells. MolPharmacol 53:62-76.). (01091 The inventors examined the ability of SR-7to alter tubulin polymerization or depolymerization in vitro. FIG. 28,Panel A. Compounds (predissolved in DMSO) were preincubated with tubulincontaining monosodium glutamate at 30 ° C for 15 min. Samples werecooled to 0 ° C and GTP was added. Samples were placed in atemperature-controlled multi-cuvette holder of a spectrophotometer heldat 0 ° C. Baselines were established and temperature was rapidly raisedto 30 ° C. Turbidity development in the cuvettes was measured at 350 nm.FIG. 28, Panel B. Compounds were added to the tubulin plus monosodiumglutamate mixture at 0 ° C, placed in the spectrophotometer andtemperature was raised to 30 ° C. Addition of 0.4 mM GTP to isolatedbovine brain tubulin produced robust polymerization that began toplateau approximately 20 min after microtubule assembly commenced (PanelA). Inclusion of 5 ,M curacin A completely inhibited GTP-induced tubulinassembly while 1 tiM curacin A caused a 50% inhibition. In contrast,SR-7 even at 40 ttM caused only moderate inhibition of tubulin assemblyas can be seen by reference to Panel A. Inhibition of 1³Hi colchicinebinding

[0102] Using methods described previously (Verdier-Pinard P, Lai J-Y,Yoo H-D, Yu J, Marquez B, Nagle DG, Nambu M, White JD, Falck JR, GerwickWH, Day BW and Hamel E (1998) Structure-activity analysis of theinteraction of curacin A, the potent colchicine site antimitotic agent,with tubulin and effects of analogs on the growth of MCF-7 breast cancercells. Mol Phannacol 53:62-76.). 5 ,uM [³H]colchicine was incubated witheither 5% DMSO vehicle or compound (5 or 50 atM) at 37° C for 15 minwith 1 UM tubulin in the presence of 1 M monosodium glutamate, 0.1 Mglucose-I-phosphate, 1 mM MgCl₂, 1 mM GTP and 0.5 mg/ml bovine serumalbumin. The solutions were filtered through two stacks ofDEAE-cellulose filters and the radioactivity in the filtrate wasdetermined by liquid scintillation spectrometry. Each series ofdeterminations included positive controls of 5 and 50 1M curacin A. Theinventors found no evidence that SR-7 could bind to the colchicine siteof tubulin. As indicated in Table III, even at 50 tM SR-7 failed tosignificantly inhibit colchicine binding while the positive control,curacin A, caused almost 90% inhibition at 5 RM. Thus, the inventorsconcluded SR-7 did not compete for the most common small molecule targeton tubulin, the colchicine binding site. The inventors propose that SR-7may act at another site such as the previously characterized vincaalkaloid site. TABLE III Percent inhibition of [³H]colchicine binding tobovine brain tubulin. PERCENT INHIBITION COMPOUND 5 μM 50 μM SR-7 1.5 ±0.5 19.6 ± 4.5 Curacin A 89.8 ± 0.6% 97.3 ± 3.1

[0103] Bcl-2 phosphorylation

[0104] Attempts to determine the phosphorylation status of Bcl-2 intsFT210 cells using two different antibodies were unsuccessful due tothe antibodies′inability to detect mouse Bcl-2 or the lack ofspecificity. Therefore, phosphorylated and non-phosphorylated Bcl-2 wasdetected in lysates from human MCF-7 cells (American Type CultureCollection, Manassas, VA) treated with microtubule perturbing agents.Equal amounts of protein were separated by electrophoresis on 15%SDS-PAGE followed by immunoblotting with an antihuman Bcl-2 antibody(sc-509, Santa Cruz Biotechnology). Positive antibody reactions werevisualized using peroxidase-conjugated secondary antibodies (JacksonImmunoResearch, West Grove, PA) and an enhanced chemiluminescencedetection system (Renaissance, NEN, Boston, MA) according tomanufacturer's instructions.

[0105] All known microtubule disrupting compounds causehyperphosphorylation ofthe antiapoptotic protein Bcl-2 (Haldar S, Jena Nand Croce CM (1995) Inactivation of bcl-2 by phospliorylation. Proc NatlAcad Sci USA 92:4507-4511 and Basu A and Haldar S (1998)Microtubule-damaging drugs trigger bcl2 phosphorylation-requirement ofphosphorylation on both serine-70 and serine-87 residues of bcl2protein. Intl J Oncol 13:659-664). Therefore, the phosphorylation statusof Bcl-2 in cells treated with various compounds including SR-7 wasexamined and the results are illustrated in FIG. 29. Proteins fromlysates of MCF-7 cells treated with 0.5 gM paclitaxel, 1 MiM nocodozole,1 ,M vincristine and, 3 or 10 HM SR-7 were separated by electrophoresison 15% SDS-PAGE followed by immunoblotting with an antibody to Bcl-2.The phosphorylated form of Bcl-2 (P) appeared as the upper bands and theunderphosphorylated form (U) was the lower band. These results wereconfirmed in a second independent experiment. Although paclitaxel,nocodazole and, vincristine caused prominent phosphorylation of Bcl-2,SR-7 at either 3 or 10 ytM failed to generate significanthyperphosphorylated Bc1-2. These results strongly suggested SR-7 did notdirectly disrupt tubulin in whole cells. f01131 The foregoingillustrations of embodiments ofthe present invention are offered for thepurposes of illustration and not limitation. It will be readily apparentto those skilled in the art that the embodiments described herein may bemodified or revised in various ways without departing from the spiritand scope of the invention. The scope of the invention is to be measuredby the appended claims.

We claim
 1. A compound of the structural formula

wherein r is a member selected from the group consisting of (e)-hc=chph,(e)-hc=chme, n- c₅h I , (e)-ch₂hc=chet, (m)-meoph, benzonitrile,2-furyl, (e,e)-hc=c(me)ch₂ch₂ch=cme₂, 3-furyl, 2-pyridyl, 3-pyridyl,4-pyridyl, and hc=ch₂:
 2. A compound of the structural formula

wherein R is a member selected from the group consisting of (p)-MeO Ph,C(Me)--CH₂, C=--H, (p)- BrPh, (p)-ClPh, 2-fuiryl(5-(p)-BrPh),2-fuiryl(5-(o)-CIPh), 2-furyl(5-(mn)-CIPh), 2-furyl(5-(o)-CF₃Ph),2-furyl(5-(m)-CF₃Ph), HC--CMe₂, (E)-C(Me)--CIBe, and (Z)C(Me)=-CHMe. 3.A method of making palmarumycin CP,, said method comprising: addingpyridinium p-toluenesulfonate to a solution of 5-hydroxy-8-methoxy- I -tetralone and ethylene glycol in benzene and heating at reflux for 30hours in a flask equipped with a Dean-Stark apparatus to produce5-hydroxy-g-methoxy-1 ,2,3 ,4-tetrahydronaphthalene-l1-spiro-2 ′-dioxolane; adding K₂C0₃ and CU₂0 to a solution of5-hydroxy-8-methoxy-1,2,3,4- tetrahydronaphthalene-l1-spiro-2′-dioxolaneand 8-iodo-l1-methoxynaphthalene, adding additional Cu₂0, treating withTsOH in a mixture of acetone/water to produce 8-methoxy-5-(8′-methoxynaphthalene-1 ′-yloxy)-3 ,4-dihydro-2H-naphthalen- 1 -one; addingBBr₃ in dichoromethane to 8-methoxy-5-(8′-methoxynaphthalene-1′-yloxy)-3,4-dihydro-2H-naphthalen-l-one to yield8-hydroxy-5-(8′-hydroxynaphthalen-1′-yloxy)- 3,4-dihydro-2H-naphthalen-1-one; reducing 8-hydroxy-5 -(8′-hydroxynaphthalen- 1′-yloxy)-3,4-dihydro-2H- naphthalen-l -one with lithium aluminum hydrideadding trifluoroethanol and Phl(OAc)₂ to produce (-8-hydroxy-1 -oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[l “,8”-de][1′,3′]dioxin; and adding Dess-Martin periodinane to()-8-hydroxy-1-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[l “,8”-de][1′,3′]dioxin indichloromethane and treating with MnO₂ in dichloromethane to producepalmarumycin CP,.
 4. A method of making deoxypreussomerin A, said methodcomprising: adding pyridinium p-toluenesulfonate to a solution of5-hydroxy-8-methoxy-I - tetralone and ethylene glycol in benzene andheating at reflux for 30 hours in a flask equipped with a Dean-Starkapparatus to produce 5-hydroxy-8-methoxy-1,2,3,4-tetrahydronaphthalene-1 -spiro-2′- dioxolane; adding K₂CO₃ andCu₂0 to a solution of 5-hydroxy-8-methoxy-1,2,3,4-tetrahydronaphthalene-l -spiro-2′-dioxolane and 8-iodo-1-methoxynaphthalene, adding additional Cu₂0, treating with TsOH in amixture of acetone/water to produce 8-methoxy-5-(8′-methoxynaphthalene-l ′-yloxy)-3,4-dihydro-2H-naphthalen-l-one; addingBBr₃ in dichoromethane to 8-methoxy-5-(8′-methoxynaphthalene-l′-yloxy)-3,4-dihydro-2H-naphthalen-l-one to yield8-hydroxy-5-(8′-hydroxynaphthalen-1′-yloxy)- 3,4-dihydro-2H-naphthalen-l-one; reducing8-hydroxy-5-(8′-hydroxynaphthalen-1′-yloxy)-3,4-dihydro-2H-naphthalen-l-one with lithium aluminum hydride adding trifluoroethanoland Phl(OAc)₂ to produce (i)-8-hydroxy-1 -oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[l “,8 ”-de] [1′,3]dioxin; adding cumene hydroperoxide and NaH to (I)-8-hydroxy-1-oxo-1,4,5,6,7,8-hexahydronaphthalene-4-spiro-2′-naphtho[1“,8”-de][1′,3′]dioxin in TUF toproduce an epoxide; and adding Dess-Martin periodinane to said epoxidein dichloromethane, treating with MnO₂ in dry dichloromethane to yielddeoxypreussomerin A.
 5. A method of making a palmarumycin CPI analogcomprising; reacting palmarymycin CP, with an alcohol of the formulaR-CH₂0H in the presence of diethyl azodicarboxylate and dichloromethane,wherein R is a member selected from the group consisting of (E)-HC=CHPh,(E)-HC=CHMe, n-C₅H,I, (E)-CH₂HC=CHEt, (m)-MeOPh, benzonitrile, 2-furyl,(E,E)-HC=C(Me)CH₂CH₂CH=CMe₂, 3 -firyl, 2-pyridyl, 3 -pyridyl, 4-pyridyl,and HC=CH₂.
 6. A method of making a palmarumycin CPI analog comprising;reacting palmarymycin CPI with an alcohol of the formula R-CH₂0H in thepresence of diethyl azodicarboxylate and dichloromethane, wherein R is amember selected from the group consisting of (p)-MeOPh, C(Me)=CH₂,C=ICH, p)-BrPh, (p)-CIPh, 2-furyl(5-(p)-BrPh), 2- furyl(5-(o)-ClPh),2-furyl(5-(m)-ClPh), 2-furyl(5-(o)-CF₃Ph), 2-faryl(5-(m)-CF₃Ph),HC=CMe₂, (E)-C(Me)=CHMe, and (Z)C(Me)=CHMe.
 7. A method of inhibitingmitosis comprising exposing cells to a compound of the structuralformula

wherein R is a member selected from the group consisting of(E)-HC--CHPh, (E)-HIC=CIIMe, n- C₅H,I, (E)-CH₂HC--CHt, (m)-MeOPh,benzonitrile, 2-furyl, (E,E)-HC--C(Me)CH2CH₂CH=CMe₂, 3-fuiryl, 2-pyrdyl,3-pyrdyl, 4-pyrdyl and HC=CH₂.
 8. The method of claim 7, wherein saidcells comprise human cancer cells.
 9. The method of claim 8, whereinsaid human cancer cells comprise breast cancer cells.
 10. The method ofclaim 9, wherein said breast cancer cells comprise MCF-7 cells,
 11. Themethod of claim 9, wherein said breast cancer cells comprise MDA-MB-23 1cells.
 12. The method of claim 8, wherein said human cancer cellscomprise ovarian cancer cells.
 13. The method of claim 12, wherein saidovarian cancer cells are a member selected from the group consisting oflA9, lA9/PTXIO and IA9/PTX22 cells.
 14. A method of inhibiting mitosiscomprising exposing cells to a compound of the structural formula

wherein R is a member selected from the group consisting of (p)-MeOPh,C(Me)=CH₂, C=CH, (p)- BrPh, (p)-ClPh, 2-furyl(5-(p)-BrPh),2-firyl(5-(o)-ClPh), 2-faryl(5-(m)-ClPh), 2-furyl(5-(o)-CF₃Ph),2-furyl(5-(m)-CF₃Ph), HC=CMe2, (E)-C(Me)=CHMe, and (Z)C(Me)=CHMe. 15.The method of claim 14, wherein said cells comprise human cancer cells.16. The method of claim 15, wherein said human cancer cells comprisebreast cancer cells.
 17. The method of claim 16, wherein said breastcancer cells comprise MCF-7 cells.
 18. The method of claim 16, whereinsaid breast cancer cells comprise MDA-MB-23 1 cells.
 19. The method ofclaim 15, wherein said human cancer cells comprise ovarian cancer cells.20. The method of claim 19, wherein said ovarian cancer cells are amember selected from the group consisting of IA9, lA9/PTXlO and1A9/PTX22 cells.
 21. A method of inhibiting proliferation of cellscomprising exposing said cells to a compound of the structural formula

wherein R is a member selected from the group consisting of (E)-HC=CHPh,(E)-HC=CHMe, n- C₅HII, (E)-CH₂HC=CHEt, (m)-MeOPh, benzonitrile, 2-furyl,(E,E)-HC=C(Me)CH₂CH₂CH=CMe₂, 3-furyl, 2-pyridyl, 3-pyridyl, 4-pyridyland HC=CH₂.
 22. The method of claim 21, wherein said cells comprisehuman cancer cells.
 23. The method of claim 22, wherein said humancancer cells comprise breast cancer cells.
 24. The method of claim 23,wherein said breast cancer cells comprise MCF-7 cells.
 25. The method ofclaim 23, wherein said breast cancer cells comprise MDA-MB-231 cells.26. The method of claim 22, wherein said human cancer cells compriseovarian cancer cells.
 27. The method of claim 26, wherein said ovariancancer cells are a member selected from the group consisting of 1 A9, IA9/PTX 10 and I A9/PTX22 cells.
 28. A method of inhibiting proliferationof cells comprising exposing said cells to a compound of the structuralformula

wherein R is a member selected from the group consisting of (p)-MeOPh,C(Me)=CH₂, C=CH, (p)- BrPh, (p)-ClPh, 2-furyl(5-(p)-BrPh),2-furyl(5-(o)-CIPh), 2-fuiryl(5-(m)-CIPh), 2-firy1(5-(o)-CF₃Ph),2-fuiryl(5-(m)-CF₃Ph), HC=CMe₂, (E)-C(Me)=CHMe, and (Z)C(Me)=CHMe. 29.The method of claim 28, wherein said cells comprise human cancer cells.30. The method of claim 29, wherein said human cancer cells comprisebreast cancer cells.
 31. The method of claim 30, wherein said breastcancer cells comprise MCF-7 cells.
 32. The method of claim 30, whereinsaid breast cancer cells comprise MDA-MB-231 cells.
 33. The method ofclaim 29, wherein said human cancer cells comprise ovarian cancer cells.34. The method of claim 33, wherein said ovarian cancer cells are amember selected from the group consisting of IA9, IA9/PTXIO and1A9/PTX22 cells.
 35. A method of inhibiting a cell′s thioredoxin-thioredoxin reductase system comprising exposing cells to a compound ofthe structural formula

wherein R is a member selected from the group consisting of (E)-HC=CHPh,(E)-HC=CHMe, n- C₅HI₁, (E)-CH₂HC=CHEt, (m)-MeOPh, benzonitrile,2-fuiryl, (E,E)-HC=C(Me)CH₂CH₂CH=CMe₂, 3-furyl, 2-pyridyl, 3-pyridyl,4-pyridyl and HC=CH₂.
 36. A method of inhibiting a cell's thioredoxin-thioredoxin reductase system comprising exposing said cells to acompound of the structural formula

wherein R is a member selected from the group consisting of (p)-MeOPh,C(Me)=CH₂, C=CH, (p)- BrPh, (p)-ClPh, 2-furyl(5-(p)-BrPh),2-furyl(5-(o)-CIPh), 2-furyl(5-(m)-ClPh), 2-fuiryl(5-(o)-CF₃Ph),2-furyl(5-(m)-CF₃Ph), HC=CMe₂, (E)-C(Me)=CHMe, and (Z)C(Me)=CHMe.
 37. Amethod of inhibiting a cell's thioredoxin - thioredoxin reductase systemcomprising exposing said cells to a compound of the structural formula

doxin - thioredoxin reductase system comprising al form-ula